Ninjurin

ABSTRACT

The present invention includes novel ninjurin proteins, peptides, and nucleic acids, as well as antibodies and pharmaceutical compositions comprising the same. The present invention further includes vectors and host cells comprising those nucleic acid sequences, chimeric protein and peptide molecules comprising the proteins and peptides of the present invention fused to heterologous polypeptide sequences. Also provided herein are antibodies which bind to the proteins and peptides of the present invention and methods for detecting the proteins and peptides of the present invention.

This invention was made with government support under grant number CA53524 awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to novel ninjurin proteins, nucleic acids andantibodies.

BACKGROUND OF THE INVENTION

A distinguishing characteristic of the peripheral nervous system (PNS),as opposed to the central nervous system (CNS), is its capacity foraxonal regeneration after injury. The environment in which PNS axonsregenerate consists of Schwann cells and their basal laminae,fibroblasts, collagen, degenerating myelin, and phagocytic cells(Fawcett, J. W. and Keynes, R. J. (1990). Annu. Rev. Neurosci. 13,43-60; Bunge, R. and Griffin, J. W. (1992). The cell of Schwann. InDiseases of the Nervous system:Clinical Neurology. A. K. Asbury, G. M.McKhann, and W. I. McDonald, eds. (Philadelphia: WB Saunders), pp.87-100). Among these components, Schwann cells are indispensable foraxonal regeneration, as evidenced by the reduction in axonal growth whenlive Schwann cells are removed from the area of injury (Hall, S. M.(1986). Neuropathol. Appl. Neurobiol. 12, 401-414). Conversely, whentransplants consisting of cultured Schwann cells and their associatedextracellular matrix are introduced into a lesion in the CNS, axonalregeneration and subsequent re-innervation is facilitated, clearlyindicating the unique role of Schwann cells in promoting axonalregeneration (Aguayo, A. J. (1985). Axonal regeneration from injuredneurons in the adult mammalian central nervous system. In Synapticplasticity and Remodeling. C. W. Cotman, ed. (New York: Guilford Press),pp. 457-483; Benfey et al., Nature 296, 150-152; Richardson et al.,(1980). Nature 284, 264-265).

The interruption of the axon following nerve injury initiates a complexseries of changes in the injured nerve. Rapid changes in the synthesisof myelin components occur, such as marked decreases in myelin lipidsynthesis (Whiten et al., (1989) J. Neurochem. 52, 1085-1092) anddiminished expression of the major myelin proteins (Trapp et al., (1988)J. Neurosci. 8, 3515-3521). Within three days after axotomy, Schwanncells in the distal stump begin to proliferate in a longitudinal bandalong which axonal regeneration and re-growth are most frequentlyobserved (Chaudhry et al, (1992) Neurologic Clinics 10, 613-627). Thereare also a number of Schwann cell proteins whose expression is increasedin the distal stump after nerve injury, including cell surface moleculeslike the p75 NGF receptor (Taniuchi et al., (1988) J. Neurosci. 8,664-681), and cell adhesion molecules LI, N-cadherin and N-CAM, that areimportant for neurite outgrowth on Schwann cells in vitro (Martini etal., (1988) J. Cell. Biol. 106, 1746; Bixby et al., (1988) J. Cell.Biol. 107, 353-361; Rieger et al., (1988) J. Cell. Biol. 107, 707-719).Schwann cells also increase the expression of a number of diffusiblemolecules, including the neurotrophic factors, brain-derivedneurotrophic factor (BDNF) and nerve growth factor (NGF), as well asapolipoprotein D, which may be involved in the recycling of cholesterolreleased from degenerating myelin (Spreyer et al, (1990) EMBO J 9,2479-2484).

Most of the changes in Schwann cell phenotype in response to nerveinjury appear to be dependent on the axon itself, as many of them arereversed as the axon regenerates. For instance, p75 expression isdown-regulated in Schwann cells adjacent to the regenerating axon(Taniuchi et al., 1988) and in Schwann cells co-cultured with neurons(Tomaselli et al., (1986) J. Cell Biol. 103, 2659-2672; Fallon, J.R.(1985) J. Neurosci. 5, 3169-3177). In contrast, the expression of myelinproteins such as P₀ and PMP-22 increases as the axon regenerates andremyelination ensues (White et al., 1989; Snipes et al., (1992) J. CellBiol. 117, 225-238). Most of the molecules that mediate alterations inSchwann cell gene expression remain obscure; one exception is theincreased expression of NGF that is mediated by IL-1 elaborated bymacrophages that invade the lesioned nerve (Lindholm et al., (1987)Nature 330, 658-659).

Cell surface adhesion proteins have also been shown to play a role intissue regeneration after injury in a number of organisms. Cell surfaceadhesion proteins also play an important role in embryonic developmentand in the assembly of adult organs. In vertebrates, a number of cellsurface glycoproteins have been identified as adhesion molecules,including integrins, cadherins, and those containing aimmunoglobulin(Ig)-like motif.

The role of adhesion proteins in nerve regeneration has been documented(Martini, R. (1994) J. Neurocytology 23, 1-28; Brodkey et al., (1993)Exp.

Neurol. 123, 251-270). The expression of a number of adhesion proteinsis elevated after nerve injury, including N-CAM and L1, which arethought to be involved in forming a suitable substrate for the extensionof the regenerating axons. The time course of this up-regulation hasbeen reported (Daniloff et al., (1986) J. Cell Biol. 103, 929-945;Martini, 1994). Interestingly, the levels of N-CAM and L1 each return tonormal more rapidly after a crush injury than after transection, inaccord with the more rapid and complete recovery observed after nervecrush (Daniloff et al., 1986). Direct support for the role of adhesionproteins in promoting neurite outgrowth has come from the demonstrationthat neutralizing antibodies to L1 and N-cadherin inhibit Schwann cellstimulated neurite outgrowth from peripheral motor neurons (Seilheimeret al., J. Cell Biol. 107, 341-351). However, these studies also suggestthat additional molecules are also important, as no single antibody orcombination of antibodies was capable of totally eliminating processoutgrowth. In the case of L1 and N-cadherin, homophilic interactionsbetween molecules present on neuronal outgrowths and Schwann cells arelikely to be responsible for their growth promoting effects (Lemmon etal., (1989) Neuron 2, 1597-1603; Takeichi, M. (1991) Science 251,1451-1455).

The residues which mediate the adhesive interactions of these moleculeshave been identified for only a subset of these proteins. One of themost well-characterized sequence motifs of this type is the tripeptideArg-Gly-Asp (RGD) which was identified as the sequence withinfibronectin that mediates cell attachment. Many integrins recognize thisRGD motif within their respective ligands, and these interactions thenmediate either cell-substratum or cell-cell interactions.

Members of the cadherin family contain multiple copies of the sequences,Asp-Arg-Glu (DRE) and Asp-x-Asn-Asp-Asn (SEQ ID NO: 1) sequences.Structural analysis of cadherin indicates that these motifs may besituated such that they can form a zipper-like structure that may becritical for cell adhesion.

Shared sequence motifs for members of the Ig-superfamily of adhesionmolecules have not been reported, although it has been proposed that adecapeptide sequence (KYSFNYDGSE) (SEQ ID NO: 2) in the third Ig-likedomain of neural cell adhesion molecule (NCAM) is responsible for itshomophilic binding interactions.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel proteins whichplay a role in axonal regeneration of PNS neuronal cells after injury.Accordingly, the invention provides recombinant ninjurin proteins andvariants thereof, and to produce useful quantities of these ninjurinproteins using recombinant DNA techniques.

It is a further object of the invention to provide recombinant nucleicacids encoding ninjurin proteins, and expression vectors and host cellscontaining the nucleic acid encoding the ninjurin protein.

An additional object of the invention is to provide polyclonal andmonoclonal antibodies directed against ninjurin proteins.

A further object of the invention is to provide methods for producingthe ninjurin proteins.

In accordance with the objects outlined above, the present inventionprovides recombinant nucleic acids encoding ninjurin proteins, includingfragments of the full-length proteins, and ninjurin peptides. Theninjurin proteins include ninjurin 1 and ninjurin 2. The ninjurinpeptides include variant or derivative ninjurin peptides.

In a further aspect, the invention provides expression vectorscomprising transcriptional and translational regulatory DNA operablylinked to DNA encoding a ninjurin protein, and host cells containing theexpression vectors.

In an additional aspect, the invention provides methods for producingninjurin proteins comprising the steps of culturing a host celltransformed with an expressing vector comprising a nucleic acid encodinga ninjurin protein and expressing the nucleic acid to produce a ninjurinprotein.

In a further aspect, the invention provides recombinant ninjurinproteins including fragments of the full-length proteins, and ninjurinpeptides, including derivative ninjurin peptides.

In an additional aspect, the invention provides pharmaceuticalcompositions comprising ninjurin proteins, and polypeptides capable ofspecifically binding to a ninjurin protein, including antibodies.

In a further aspect, the invention provides polyclonal or monoclonalantibodies to ninjurin proteins.

In an additional aspect, the invention provides METHODS OF TREATMENT,ETC.?

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the nucleotide sequence (FIG. 1A) (SEQ ID NO: 3)and amino acid sequence (FIG. 1B) (SEQ ID NO: 4) of rat ninjurin 1.

FIGS. 2A and 2B depict the nucleotide sequence (FIG. 2A) (SEQ ID NO: 5)and amino acid sequence (FIG. 2B) (SEQ ID NO: 6) of human ninjurin 1.

FIGS. 3A and 3B depict the nucleotide sequence (FIG. 3A) (SEQ ID NO: 7)and amino acid sequence (FIG. 3B) (SEQ ID NO: 8) of human ninjurin 2.

FIG. 4 depicts the nucleotide sequence (SEQ ID NO: 9) of variant 1 ofhuman ninjurin 1.

FIG. 5 depicts the nucleotide sequence (SEQ ID NO: 10) of variant 1 ofhuman ninjurin 2.

FIG. 6 depicts the nucleotide sequence (SEQ ID NO: 11) of variant 2 ofhuman ninjurin 2.

FIG. 7 depicts the homology lineup of rat ninjurin 1 (SEQ ID NO: 4),human ninjurin 1 (SEQ ID NO: 6), and human ninjurin 2 (SEQ ID NO: 8).

FIGS. 8A, 8B and 8C show the peptides used to characterize the bindingdomain. FIG. 8A depicts the first 100 amino acids of the rat ninjurin 1protein (SEQ ID NO: 13), with the overlapping peptides used to elucidatethe binding domain. The double underlining shows the putativetransmembrane domain. FIG. 8B depicts the 8 overlapping peptides used tolocalize the binding domain. The "+" or "-" next to the peptide showsthe peptide's ability to inhibit the ninjurin-mediated aggregation. FIG.8C shows the 7 peptides (SEQ ID NO: 14-20) used to identify theimportant binding residues. The single amino acid substitutions used toidentify the important binding domain residues are shown in bold.Peptides 9 (SEQ ID NO: 14) (substitution of Arg28 (rat ninjurin 1numbering) to Asn28), 10 (SEQ ID NO: 15) (substitution of Trp29 forAla29), 13 (SEQ ID NO: 18) (substitution of Arg32 for Asn32), and 15(SEQ ID NO: 20) (substitution of Arg34 for Asn34) were less active thanpeptide 6 for inhibition of adhesion, thus implicating these residues asfunctionally important for ninjurin adhesion. Peptides 11 (SEQ ID NO:16) (substitution of Gly29 to Ala29), 12 (SEQ ID NO: 17) (substitutionof Leu3 1 to Asn3 1) did not exhibit altered inhibition, thus suggestingthese residues are not important in ninjurin binding. Peptide 14 (SEQ IDNO: 19) (substitution of Asn33 for Leu33) was actually more effectivethan the wild type peptide 6 in inhibiting ninjurin binding.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel ninjurin proteins. In a preferredembodiment, the ninjurin proteins are from vertebrates, more preferablyfrom mammals, and in the preferred embodiment, from rats or humans.However, using the techniques outlined below, ninjurin proteins fromother organisms may also be obtained.

As outlined herein, the ninjurin proteins of the present invention arecellular adhesion molecules which are expressed in a variety of tissuetypes. Ninjurin is a membrane bound protein, as it contains two putativetransmembrane domains, with the extracellular domain containing at leastone binding domain. The mechanism of adhesion includes (but is notlimited to) a homophilic binding of ninjurin molecules on adjacentcells, i.e. the binding domain of ninjurin can self-aggregate. It isalso possible that the mechanism of adhesion includes heterophilicinteractions.

Without being bound by theory, it appears that there are a number ofrelated ninjurin proteins and nucleic acids. For example, in the human,it appears that there are at least two separate ninjurin nucleic acidsthat encode two different ninjurin proteins, ninjurin 1 and ninjurin 2.In addition, the ninjurin nucleic acids also are alternatively splicedto produce further distinct ninjurin nucleic acids or variant ninjurinnucleic acids such as are depicted in FIGS. 4 (SEQ ID NO: 9), 5 (SEQ IDNO: 10) and 6 (SEQ ID NO: 11). All of these ninjurins share basichomology to each other, and a marked lack of homology to any other knownproteins or nucleic acids.

Thus, a ninjurin protein of the present invention may be identified inseveral ways. A ninjurin nucleic acid or ninjurin protein is initiallyidentified by substantial nucleic acid and/or amino acid sequencehomology to the sequences shown in FIGS. 1 (SEQ ID NOS: 3 and 4), 2 (SEQID NOS: 5 and 6), 3 (SEQ ID NOS: 7 and 8), 4 (SEQ ID NO: 9), 5 (SEQ IDNO: 10) or 6 (SEQ ID NO: 11). Such homology can be based upon theoverall nucleic acid or amino acid sequence.

As used herein, a protein is a "ninjurin protein" if the overallhomology of the protein sequence to the amino acid sequences shown inFIGS. 1 (SEQ ID NOS: 3 and 4), 2 (SEQ ID NOS: 5 and 6) or 3 (SEQ ID NOS:7 and 8) is preferably greater than about 40%, more preferably greaterthan about 50% and most preferably greater than 75%. In some embodimentsthe homology will be as high as about 90 to 95 or 98%. This homologywill be determined using standard techniques known in the art, such asthe Best Fit sequence program described by Devereux et al., NucL. AcidRes. 12:387-395 (1984) or the BLASTX program (Altschul et al., J. Mol.Biol. 215, 403-410). The alignment may include the introduction of gapsin the sequences to be aligned. In addition, for sequences which containeither more or fewer amino acids than the proteins shown in the Figures,it is understood that the percentage of homology will be determinedbased on the number of homologous amino acids in relation to the totalnumber of amino acids. Thus, for example, homology of sequences shorterthan that shown in the Figures, as discussed below, will be determinedusing the number of amino acids in the shorter sequence.

As discussed above, ninjurin proteins include ninjurin 1 and ninjurin 2proteins. Ninjurin proteins may be identified in one aspect bysignificant homology to at least one and preferably both of thetransmembrane domains, as these domains are quite unique. Thus, aninjurin 1 protein is characterized by homology to the rat and humanninjurin 1 proteins depicted in FIGS. 1B (SEQ ID NO: 4) and 2B (SEQ IDNO: 6). This homology is preferably greater than about 50%, with greaterthan about 70% being particularly preferred and greater than about 85%being especially preferred. In some cases the homology will be greaterthan about 90 to 95 or 98%. As discussed herein, the rat and humanninjurin 1 proteins are roughly 90% homologous to each other.Preferably, a ninjurin 1 protein also has significant homology to theninjurin 1 binding domain as described herein. Similarly, a ninjurin 2protein is characterized by homology to the human ninjurin 2 protein.This homology is preferably greater than about 50%, with greater thanabout 70% being particularly preferred and greater than about 85% beingespecially preferred. In some cases the homology will be greater thanabout 90 to 95 or 98%. Ninjurin 1 and 2 proteins are homologous to eachother, with the ninjurin 1 and 2 proteins from human being roughly 55%homologous to each other.

Ninjurin proteins of the present invention may be shorter or longer thanthe amino acid sequences shown in the Figures. Thus, in a preferredembodiment, included within the definition of ninjurin proteins areportions or fragments of the sequences shown in FIGS. 1 (SEQ ID NOS: 3and 4), 2 (SEQ ID NOS: 5 and 6) and 3 (SEQ ID NOS: 7 and 8). As outlinedin the Examples, ninjurin peptides can be made which comprise theninjurin 1 binding domain of rat ninjurin and human ninjurin 1. Theseninjurin peptides will bind to the binding domain of the full lengthprotein, or to each other, thus preventing or decreasing their abilityto bind other binding domains, and thus interfering with cellularadhesion. Alternatively, longer fragments of ninjurin proteins can bemade, or fragments of ninjurin which do not contain the binding domain.A preferred ninjurin fragment is the extracellular domain of ninjurin,comprising roughly the first 70 amino acids of ninjurin I or the aboutthe first 55 amino acids of ninjurin 2.

Furthermore, as outlined in the Examples, there are at least twodistinct human ninjurin genes, encoding two ninjurin proteins. Inaddition, the mRNA for these two genes can be alternatively spliced.These alternatively spliced forms are also included with the definitionof a ninjurin nucleic acid, despite the lack of significant amino acidsequence and a ninjurin 1 binding domain in the putative coding region.

Thus, in a preferred embodiment, the ninjurin proteins of the presentinvention are ninjurin peptides. In this embodiment, a ninjurin peptidecomprises at least the ninjurin 1 binding domain, although it maycontain additional amino acids as well. As shown in the Examples anddiscussed below, ninjurin is a homophilic adhesion protein. A "bindingdomain", i.e. a portion of the protein that associates during cellularaggregation, will bind to other ninjurin binding domains. The ninjurin 1binding domain for the rat and human ninjurin 1 protein has beenidentified as shown in the Examples. The ninjurin 1 binding domainincludes the amino acid residues corresponding to residues 26-37 of therat or human ninjurin 1, with at least residues corresponding to aminoacids numbers 28-35 preferably being present. The rat and human ninjurin1 sequences are 70% homologous as between residues 26-37, and 57%homologous as between residues 2-35. Thus, in a preferred embodiment, aninjurin protein or peptide may be identified by the presence of abinding domain. A ninjurin 1 protein or peptide contains a ninjurin 1binding domain.

A ninjurin 2 protein or peptide may contain a ninjurin 2 binding domain.In a preferred embodiment, the binding domain is at least about 50%homologous to any of the binding domains depicted in FIG. 7 (SEQ ID NO:4,6,8 and 12), with at least about 60% being preferred, and at leastabout 70% being particularly preferred. In some embodiments the homologywill be as high as about 85% or 90%, with 95% and 98% being particularlypreferred.

Thus, a ninjurin peptide is a short protein that contains a homologousbinding domain as outlined above. The ninjurin peptides are preferablyat least about 7 or 10 to 15 amino acids long, although longer peptideswhich include all or part of the binding domain may also be used, aswell as other amino acid residues if desirable. It should be appreciatedthat the exact length of the ninjurin peptide may vary. Preferredembodiments of ninjurin 1 peptides include, but are not limited to,(from rat ninjurin 1) Pro-Pro-Arg-Trp-Gly-Leu-Arg-Asn-Arg-Pro-Ile-Asn(SEQ ID NO: 21), Pro-Arg-Trp-Gly-Leu-Arg-Asn-Arg-Pro (SEQ ID NO: 22),and Arg-Trp-Gly-Leu-Arg-Asn-Arg (SEQ ID NO: 23). Preferred ninjurin 1peptides from human ninjurin 1 include, but are not limited to,Pro-Ala-Arg-Trp-Gly-Trp-Arg-His-Gly-Pro-Ile-Asn (SEQ ID NO: 24),Ala-Arg-Trp-Gly-Trp-Arg-His-Gly-Pro (SEQ ID NO: 25),Arg-Trp-Gly-Trp-Arg-His (SEQ ID NO: 26), and Arg-Trp-Gly-Trp-Arg (SEQ IDNO: 27).

However, not all ninjurin proteins contain a complete ninjurin 1 bindingdomain. For example, hunan ninjurin 2 does not appear to contain acomplete ninjuurin 1 binding domain. In addition, it appears that thereare variant forms of the ninjurin genes that do not contain ninjurin 1binding domains. Ninjurin 2 proteins may contain an as yet unidentifiedninjurin 2 binding domain. The ninjurin 2 binding domain may be easilyidentified by those skilled in the art as is outlined in the Examplesfor ninjurin 1. For example, overlapping peptides of ninjurin 2 are madeand tested in ninjurin mediated binding assays. Peptides which inhibitninjurin mediated binding contain all or portion of a ninjurin 2 bindingdomain. The ninjurin 2 binding domain may be further identified usingsite directed mutagenesis as outlined in Example 2.

In a preferred embodiment, the ninjurin peptides are derivative orvariant ninjurin peptides, either of ninjurin 1 or 2 or others. That is,the derivative ninjurin peptide will contain at least one amino acidsubstitution, deletion or insertion, with amino acid substitutions beingparticularly preferred. The amino acid substitution, insertion ordeletion may occur at any residue within the ninjurin peptide. Asoutlined below, particularly preferred substitutions are made atpositions corresponding to rat ninjurin 1 at position 28, 29, 31, 32, 33and 34. The derivative ninjurin peptide can be less effective or moreeffective in binding to another binding domain of either anotherninjurin peptide or a full length ninjurin protein, or may not alter thebinding characteristics. By "less effective" herein is meant that themutation comprising the derivative peptide makes the peptide less activefor inhibition of ninjurin mediated adhesion than the wild-type ninjurinpeptide. By "more effective" herein is meant that the derivative peptideis more active for inhibition of ninjurin mediated adhesion than thewild-type ninjurin peptide, i.e. the derivative binds the binding domainmore tightly.

Ninjurin proteins may also be identified as being encoded by ninjurinnucleic acids. Thus, ninjurin proteins are encoded by nucleic acids thatwill hybridize to the sequences depicted in FIGS. 1-6 (SEQ ID NO: 3-11),as outlined herein.

In a preferred embodiment, when the ninjurin protein is to be used togenerate antibodies, the ninjurin protein must share at least oneepitope or determinant with the full length proteins shown in FIGS. 1(SEQ ID NOS: 3 and 4), 2 (SEQ ID NOS: 5 and 6), 3 (SEQ ID NOS: 7 and 8),4 (SEQ ID NO: 9), 5 (SEQ ID NO: 10), and 6 (SEQ ID NO: 11). By "epitope"or "determinant" herein is meant a portion of a protein which willgenerate and/or bind an antibody. Thus, in most instances, antibodiesmade to a smaller ninjurin protein will be able to bind to the fulllength protein. In a preferred embodiment, the epitope is unique; thatis, antibodies generated to a unique epitope show little or nocross-reactivity. In a preferred embodiment, the antibodies aregenerated to a ninjurin binding domain. In some instances, the antibodymay recognize both ninjurin 1 and 2 molecules; alternatively, antibodiesmay be generated that do not cross-react as between ninjurin 1 andninjurin 2. In a preferred embodiment, the antibodies are generated toan extracellular portion of the ninjurin molecule, i.e. to all or someof the N-terminal region from amino acid numbers 1-71. The ninjurinantibodies of the invention specifically bind to ninjurin proteins. By"specifically bind" herein is meant that the antibodies bind to theprotein with a binding constant in the range of at least 10⁴ -10⁶ M⁻¹,with a preferred range being 10⁷ -10⁹ M⁻¹.

In the case of the nucleic acid, the overall homology of the nucleicacid sequence is commensurate with amino acid homology but takes intoaccount the degeneracy in the genetic code and codon bias of differentorganisms.

Accordingly, the nucleic acid sequence homology may be either lower orhigher than that of the protein sequence. Thus the homology of thenucleic acid sequence as compared to the nucleic acid sequences of FIGS.1 (SEQ ID NOS: 3 and 4), 2 (SEQ ID NOS: 5 and 6), 3 (SEQ ID NOS: 7 and8), 4 (SEQ ID NO. 9), 5 (SEQ ID NO: 10) or 6 (SEQ ID NO: 11) ispreferably greater than 40%, more preferably greater than about 45%,particularly greater than about 50% and most preferably greater than55%. In some embodiments the homology will be as high as about 70, 80,90 to 95 or 98%. The homology of the nucleic acid sequence of ratninjurin 1 and human ninjurin 1 is 73%; as between rat ninjurin 1 andhuman ninjurin 2 the homology is 52%; and as between human ninjurin 1and 2 the homology is 55%.

In a preferred embodiment, a ninjurin nucleic acid encodes a ninjurinprotein; ninjurin 1 nucleic acids encode ninjurin 1 proteins, andninjurin 2 nucleic acids encode ninjurin 2 proteins. As will beappreciated by those in the art, due to the degeneracy of the geneticcode, an extremely large number of nucleic acids may be made, all ofwhich encode the ninjurin proteins of the present invention. Thus,having identified a particular amino acid sequence, those skilled in theart could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the ninjurin.

In one embodiment, the nucleic acid homology is determined throughhybridization studies. Thus, for example, nucleic acids which hybridizeunder high stringency to the nucleic acid sequences shown in FIGS. 1-6or their complements are considered ninjurin genes. High stringencyconditions are known in the art; see for example Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra.

The ninjurin proteins and nucleic acids of the present invention arepreferably recombinant. As used herein, "nucleic acid" may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA andoligonucleotides including sense and anti-sense nucleic acids. Suchnucleic acids may also contain modifications in the ribose-phosphatebackbone to increase stability and half life of such molecules inphysiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. By theterm "recombinant nucleic acid" herein is meant nucleic acid, originallyformed in vitro, in general, by the manipulation of nucleic acid byendonucleases, in a form not normally found in nature. Thus an isolatedninjurin nucleic acid, in a linear form, or an expression vector formedin vitro by ligating DNA molecules that are not normally joined, areboth considered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention.

Similarly, a "recombinant protein" is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. The definition includes the production of a ninjurin proteinfrom one organism in a different organism or host cell. Alternatively,the protein may be made at a significantly higher concentration than isnormally seen, through the use of a inducible promoter or highexpression promoter, such that the protein is made at increasedconcentration levels. Alternatively, the protein may be in a form notnormally found in nature, as in the addition of an epitope tag or aminoacid substitutions, insertions and deletions, as discussed below.

Also included with the definition of ninjurin protein are other ninjurinproteins of the ninjurin family, and ninjurin proteins from otherorganisms, which are cloned and expressed as outlined below. Thus, probeor degenerate polymerase chain reaction (PCR) primer sequences may beused to find other related ninjurin proteins from humans or otherorganisms. As will be appreciated by those in the art, particularlyuseful probe and/or PCR primer sequences include the unique areas of theninjurin nucleic acid sequence. Thus, useful probe or primer sequencesmay be designed to: a) all or part of the sequence of the uniqueninjurin 1 binding domain; b) all or part of the unique first putativetransmembrane domain, which spans roughly amino acids 72 to 100 in ratand human ninjurin 1 and amino acids 59 to 87 in human ninjurin 2, withresidues 70-80 (ninjurin 1) and 57-67 (ninjurin 2) being particularlyuseful due to the presence of phenylalanine and tryptophan; c) all orpart of the unique second putative transmembrane domain, which spansroughly amino acids 118-139 in rat and human ninjurin 1 and amino acids105-126 in human ninjurin 2; and d) all or part of the residues fromabout 50 to about 70 in ninjurin 1 and 37-77 of ninjurin 2, due to thepresence of conserved methionines. As is generally known in the art,preferred PCR primers are from about 15 to about 35 nucleotides inlength, with from about 20 to about 30 being preferred, and may containinosine as needed. The conditions for the PCR reaction are well known inthe art.

Once the ninjurin nucleic acid is identified, it can be cloned and, ifnecessary, its constituent parts recombined to form the entire ninjurinprotein nucleic acid. Once isolated from its natural source, e.g.,contained within a plasmid or other vector or excised therefrom as alinear nucleic acid segment, the recombinant ninjurin nucleic acid canbe further used as a probe to identify and isolate other ninjurinnucleic acids. It can also be used as a "precursor" nucleic acid to makemodified or variant ninjurin nucleic acids and proteins.

Using the nucleic acids of the present invention which encode a ninjurinprotein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the ninjurin protein."Operably linked" in this context means that the transcriptional andtranslational regulatory DNA is positioned relative to the codingsequence of the ninjurin protein in such a manner that transcription isinitiated. Generally, this will mean that the promoter andtranscriptional initiation or start sequences are positioned 5' to theninjurin protein coding region. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the ninjurin protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the ninjurin protein in Bacillus. Numeroustypes of appropriate expression vectors, and suitable regulatorysequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

The ninjurin proteins of the present invention are produced by culturinga host cell transformed with an expression vector containing nucleicacid encoding a ninjurin protein, under the appropriate conditions toinduce or cause expression of the ninjurin protein. The conditionsappropriate for ninjurin protein expression will vary with the choice ofthe expression vector and the host cell, and will be easily ascertainedby one skilled in the art through routine experimentation. For example,the use of constitutive promoters in the expression vector will requireoptimizing the growth and proliferation of the host cell, while the useof an inducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of the harvestis important. For example, the baculoviral systems used in insect cellexpression are lytic viruses, and thus harvest time selection can becrucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melangaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanomacell lines, and immortalized mammalian myeloid, lymphoid cell lines.

In a preferred embodiment, the ninjurin proteins are expressed inmammalian cells. Mammalian expression systems are also known in the art.A mammalian promoter is any DNA sequence capable of binding mammalianRNA polymerase and initiating the downstream (3') transcription of acoding sequence for ninjurin protein into mRNA. A promoter will have atranscription initiating region, which is usually placed proximal to the5' end of the coding sequence, and a TATA box, using a located 25-30base pairs upstream of the transcription initiation site. The TATA boxis thought to direct RNA polymerase II to begin RNA synthesis at thecorrect site. A mammalian promoter will also contain an upstreampromoter element (enhancer element), typically located within 100 to 200base pairs upstream of the TATA box. An upstream promoter elementdetermines the rate at which transcription is initiated and can act ineither orientation. Of particular use as mammalian promoters are thepromoters from mammalian viral genes, since the viral genes are oftenhighly expressed and have a broad host range. Examples include the SV40early promoter, mouse mammary tumor virus LTR promoter, adenovirus majorlate promoter, herpes simplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3' to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3' terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenlytion signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, ninjurin proteins are expressed in bacterialsystems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3')transcription of the coding sequence of ninjurin protein into MRNA. Abacterial promoter has a transcription initiation region which isusually placed proximal to the 5' end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site. Sequences encodingmetabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgamo (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the ninjurin protein in bacteria. The signalsequence typically encodes a signal peptide comprised of hydrophobicamino acids which direct the secretion of the protein from the cell, asis well known in the art. The protein is either secreted into the growthmedia (gram-positive bacteria) or into the periplasmic space, locatedbetween the inner and outer membrane of the cell (gram-negativebacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, ninjurin proteins are produced in insect cells.Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

In a preferred embodiment, ninjurin protein is produced in yeast cells.Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred promoter sequences for expression inyeast include the inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUPI gene, which allows yeast to grow in the presence of copperions.

The ninjurin protein may also be made as a fusion protein, usingtechniques well known in the art. Thus, for example, for the creation ofmonoclonal antibodies, if the desired epitope is small, the ninjurinprotein may be fused to a carrier protein to form an immunogen.Alternatively, the ninjurin protein may be made as a fusion protein toincrease expression, or for other reasons. For example, when theninjurin protein is a ninjurin peptide, the nucleic acid encoding thepeptide may be linked to other nucleic acid for expression purposes.

Also included within the definition of ninjurin proteins of the presentinvention are amino acid sequence variants. These variants fall into oneor more of three classes: substitutional, insertional or deletionalvariants. These variants ordinarily are prepared by site specificmutagenesis of nucleotides in the DNA encoding the ninjurin protein,using cassette or PCR mutagenesis or other techniques well known in theart, to produce DNA encoding the variant, and thereafter expressing theDNA in recombinant cell culture as outlined above. However, variantninjurin protein fragments having up to about 100-150 residues may beprepared by in vitro synthesis using established techniques. Amino acidsequence variants are characterized by the predetermined nature of thevariation, a feature that sets them apart from naturally occurringallelic or interspecies variation of the ninjurin protein amino acidsequence. The variants typically exhibit the same qualitative biologicalactivity as the naturally occurring analogue, although variants can alsobe selected which have modified characteristics as will be more fullyoutlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed ninjurin variants screened forthe optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of ninjurinprotein activities; for example, for binding domain mutations,competitive binding studies such as are outlined in the Examples may bedone.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger. For example, a preferred deletion variant is the deletionof the transmembrane domains and the cytoplasmic domain, leaving onlythe extracellular domain of ninjurin, i.e. a soluble receptor. Theextracellular domain of ninjurin 1 comprises the N-terminal 70 or soamino acids, and the extracellular domain of ninjurin 2 comprises theN-terminal 57 or so amino acids.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the ninjurin protein aredesired, substitutions are generally made in accordance with thefollowing chart:

    ______________________________________                                        Chart I                                                                             Original Residue                                                                              Exemplary Substitutions                                 ______________________________________                                        Ala               Ser                                                           Arg Lys                                                                       Asn Gln, His                                                                  Asp Glu                                                                       Cys Ser                                                                       Gln Asn                                                                       Glu Asp                                                                       Gly Pro                                                                       His Asn, Gln                                                                  Ile Leu, Val                                                                  Leu Ile, Val                                                                  Lys Arg, Gln, Glu                                                             Met Leu, Ile                                                                  Phe Met, Leu, Tyr                                                             Ser Thr                                                                       Thr Ser                                                                       Trp Tyr                                                                       Tyr Trp, Phe                                                                  Val Ile, Leu                                                                ______________________________________                                    

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the ninjurin proteins as needed. Alternatively, thevariant may be designed such that the biological activity of theninjurin protein is altered. For example, the N-glycosylation site maybe altered or removed. Either or both of the transmembrane domains maybe altered or removed, to make a soluble or secreted protein, i.e. theextracellular domain.

In a preferred embodiment, binding domain variants are made. In oneembodiment, a binding domain may be eliminated entirely. Alternatively,any or all of the amino acids of a binding domain may be be altered ordeleted. In a preferred embodiment, one or more of the amino acids ofthe binding domain are substituted by other amino acids. Thus, aminoacids corresponding to the rat ninjurin 1 binding domain residues may bealtered, including, but not limited to, residues corresponding to Pro26,Pro27, Arg28, Trp29, Gly3O, Leu31, Arg32, Asn33, Arg34, Pro35, Ile36 orAsn37. Particularly preferred are alterations at Arg28, Trp29, Arg32,Asn33 and Arg34, alone or in combination. Any amino acid may besubstituted. In particular, modifications at position 33 can increasethe binding affinity of the molecule. Similarly, the amino acids of thehuman ninjurin 1 binding domain may be altered, including, but notlimited to, residues corresponding to Pro26, Ala27, Arg28, Trp29, Gly3O,Trp31, Arg32, His33, Gly34, Pro35, Ile36, or Asn37, alone or incombination, with alterations at Arg28, Trp29, Trp31, Arg32 and His33being particularly preferred. Again, any amino acid may be substituted.Similarly, the residues within the putative ninjurin 1 binding domain ofhuman ninjurin 2 may be altered, including residues corresponding toArg32, Ser33, Gln34, Pro35, Ile36 and Asn37 (rat ninjurin 1 numbering).Furthermore, human ninjurin 2 protein may be altered to contain acomplete ninjurin 1 binding domain.

In addition, as outlined in the Examples, the homophilic ninjurin 1cellular adhesion is dependent on the presence of divalent cations;thus, the metal binding properties of the binding domain may be altered.

In one embodiment, the ninjurin nucleic acids, proteins and antibodiesof the invention are labelled. By "labelled" herein is meant that acompound has at least one element, isotope or chemical compound attachedto enable the detection of the compound. In general, labels fall intothree classes: a) isotopic labels, which may be radioactive or heavyisotopes; b) immune labels, which may be antibodies or antigens; and c)colored or fluorescent dyes. The labels may be incorporated into thecompound at any position.

In a preferred embodiment, the ninjurin protein is purified or isolatedafter expression. Ninjurin proteins may be isolated or purified in avariety of ways known to those skilled in the art depending on whatother components are present in the sample. Standard purificationmethods include electrophoretic, molecular, immunological andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the ninjurin protein may be purified using a standardanti-ninjurin antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.For general guidance in suitable purification techniques, see Scopes,R., Protein Purification, Springer-Verlag, NY (1982). The degree ofpurification necessary will vary depending on the use of the ninjurinprotein. In some instances no purification will be necessary.

Once expressed and purified if necessary, the ninjurin proteins areuseful in a number of applications.

In a preferred embodiment, the ninjurin proteins, and particularlyninjurin peptides, are useful in the study or treatment or otherconditions which are mediated by ninjurin, i.e. to treat or preventninjurin-mediated disorders. Thus, "ninjurin mediated disorders" includeconditions involving inappropriate (i.e. excessive or insufficient)cellular adhesion. Undesirable (i.e. excessive) cellular adhesion hasbeen implicated in a large number of conditions, including, but notlimited to, inflammatory diseases such as rheumatoid arthritis, asthma,allergy conditions, adult respiratory distress syndrome, inflammatorybowel diseases (e.g. Crohn's disease, ulcerative colitis and regionalenteritis) and opthalmic inflammatory diseases; autoimmune diseases;thrombosis or inappropriate platelet aggregation conditions;arteriosclerosis; reocculusion following thrombolysis; cardiovasculardiseases; some forms of diabetes and neoplastic disease includingmetastasis conditions. See generally WO 93/08823 and WO 95/17412, herebyincorporated by reference, which discuss the role of other cellularadhesion molecules in a number of diseases, which may also be mediatedby ninjurin.

Thus, in a preferred embodiment, ninjurin proteins or peptidescomprising the binding domain are made as "blocking peptides" for thetreatment of undesirable cellular adhesion. These peptides can be usedas competitive binding proteins for ninjurin mediated adhesion, thuseffectively decreasing or blocking adhesion. Thus, any physiologicalprocess that depends on ninjurin mediated adhesion for biologicalfinction may be altered by the use of ninjurin blocking peptides orninjurin proteins. For example, adhesion events in inflammatoryresponses are known, including cell migration of macrophages to the siteof inflammation, which may be blocked by the addition of competingbinding domains, either as peptides, larger fragments or the full lengthproteins. Similarly, cell adhesion also contributes to metastasis ofcancerous tumors, and blocking peptides could be used which wouldprevent cellular adhesion of metastasizing tumor cells. Furthermore,conditions which have active vascularization, such as in rapidly growingtumor cells, and diabetic retinopathy may rely on ninjurin mediatedadhesion for neovascularization, and may be treated or altered via theuse of blocking peptides. Additionally, harmful blood clotting may alsobe caused by inappropriate cell adhesion, particularly cell adhesion tothe extracellular matrix, such as in arteriosclerosis, which could beprevented by the administration of a ninjurin peptide or protein thatwould function as an antagonist. Thus, proteins or polypeptides thatspecifically bind to ninjurin proteins may be made and used, which maybe ninjurin proteins, including fragments of ninjurin proteins andninjurin peptides, as well as ninjurin antibodies, discussed below.

Alternatively, cellular adhesion may also be desirable in someapplications. Thus, for example, in a preferred embodiment, ninjurinproteins or peptides are used to promote nerve regeneration. Forexample, ninjurin nucleic acids may be used to transform cells whichthen express ninjurin at a high density at the surface. The cells maythen be transplanted into a site of nerve damage to promote nerveregeneration. Similarly, ninjurin proteins or peptides may be made andincorporated into a matrix such as a gel matrix to promote nerveregeneration. Additionally, wound healing may be prolonged when cellularadhesion is insufficient; a ninjurin protein or peptide may be attachedto a surface, matrix or cell and used to promote wound healing.Similarly, in prosthetic implantation, coating the prosthesis withninjurin proteins would promote cellular adhesion and minimizerejection.

In one embodiment, the ninjurin proteins of the present invention may beused to generate polyclonal and monoclonal antibodies to ninjurinproteins, which are useful as described below. Similarly, the ninjurinproteins can be coupled, using standard technology, to affinitychromatography columns. These columns may then be used to purifyninjurin antibodies. In a preferred embodiment, the antibodies aregenerated to epitopes unique to the ninjurin protein; that is, theantibodies show little or no cross-reactivity to other proteins. Theseantibodies find use in a number of applications. For example, theninjurin antibodies may be coupled to standard affinity chromatographycolumns and used to purify ninjurin proteins. The antibodies may also beused as blocking polypeptides, as outlined above, since they willspecifically bind to the ninjurin protein.

The polypeptides that specifically bind to ninjurin, i.e. ninjurinproteins and ninjurin antibodies, may be used in the diagnosis ofninjurin-mediated disorders. Thus, the presence or absence or ninjurinmay be assayed or detected using labelled ninjurin proteins, antibodiesor nucleic acids. For example, methods are provided for detecting aninjurin protein in a target sample comprising contacting a labelledpolypeptide which will specifically bind to a ninjurin protein with thetarget sample and assaying for the presence of binding between thelabelled polypeptide and ninjurin, if present, in the target sample. Thecontacting is done under conditions which allow binding to ninjurin.Thus, lymphocytes may be screened for the presence or absence ofninjurin.

The ninjurin proteins herein also find use as a target in screeningassays for the development of ninjurin antagonists.

In the preferred embodiment, the ninjurin proteins of the presentinvention are administered to a patient to mediate ninjurin relatedadhesion, as outlined above.

In this embodiment, a therapeutically effective dose of a ninjurin isadministered to a patient. By "therapeutically effective dose" herein ismeant a dose that produces the effects for which it is administered. Theexact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for ninjurin degradation, systemic versuslocalized delivery, and rate of new protease synthesis, as well as theage, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

A "patient" for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The administration of the ninjurin proteins of the present invention canbe done in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the ninjurin may be directly applied as asolution or spray.

The pharmaceutical compositions of the present invention comprise aninjurin in a form suitable for administration to a patient. In thepreferred embodiment, the pharmaceutical compositions are in a watersoluble form, such as being present as pharmaceutically acceptablesalts, which is meant to include both acid and base addition salts."Pharmaceutically acceptable acid addition salt" refers to those saltsthat retain the biological effectiveness of the free bases and that arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. "Pharmaceutically acceptable base addition salts" include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLES Example 1 Cloning and characterization of rat ninjurin

Animals and surgical procedures. All surgical procedures followed theNIH guidelines for care and use of laboratory animals at WashingtonUniversity. Male Sprague-Dawley rats (200-300 g) were anesthetized, andthe right sciatic nerve was injured at the hip level either bytransection or by compressing the nerve with forceps for 30 sec (crush).The contralateral nerve was exposed, but left uninjured (control). Afterthe indicated length of time, the animals were euthanized and eitherperfused with 4% paraformaldehyde/phosphate buffered saline (PBS) forimmunohistochemical analysis or decapitated for immediate collection oftissues for RNA isolation or in situ hybridization analysis. For nerveligation experiments, the nerve was tightly ligated at the level of thehip using a 4-0 nylon suture. When indicated, the nerve was transected5-8 mm distal to the ligation site. For analysis of E17 and E19 embryos,timed pregnant rats of the indicated gestational age were sacrificed andthe embryos were immediately frozen. All tissue samples were stored at-70 C.

Construction and Screening of Sciatic Nerve cDNA libraries. To identifygenes regulated in Schwann cells by nerve injury, two rat sciatic nervecDNA libraries (normal vs. injured) were constructed in the λZAPIIvector (Stratagene). The first was made using poly[A⁺ ]RNA isolated fromnormal sciatic nerve, whereas the other was made using equivalentamounts of poly[A⁺ ]RNA isolated from 16 hr, 3 d and 7 d post-axotomynerve segments (both proximal neuroma and distal segment). The RNAtemplates were used to synthesize cDNA as previously described (Gubleret al., (1983) Gene 25, 263-269). The cDNAs were cloned into EcoR1/Not1digested λZAPII phage arms, the phage were packaged in vitro (GigapackGold; Stratagene) and used to infect E. coli XL-1 Blue strain(Stratagene). To enrich for cDNA clones whose mRNAs are upregulated inresponse to axotomy, a subtracted injured nerve library was obtainedusing a minor modification of a previously described method (Rubensteinet al., (1990) Nucleic Acids Res 18, 4833-4842). RNA was transcribedfrom the normal nerve cDNA library and biotinylated using Photobiotin(Vector Laboratories, CA). This biotinylated RNA was hybridized tosingle-stranded phagemid prepared from the injured nerve library andheteroduplexes and unhybridized driver RNA was removed with streptavidinSepharose beads (Sigma). The remaining single-stranded phagemid wasconverted to double-stranded form with Taq polymerase and transformedinto XL-1 Blue bacteria. This subtracted library was differentiallyscreened by hybridizing with ³² P-labeled first strand cDNAs prepared byreverse transcription of poly[A⁺ ]RNA extracted from either distalportion of the transected sciatic nerve (7 days after transection) orsciatic nerves of the contralateral side (control) as previouslydescribed (Maniatis et al.,(1982). Molecular cloning: A laboratorymanual. Cold Spring Harbor Laboratory 1, 280-281). Plaques that gave asignal of differential intensity between the two probes were re-platedand re-screened with the same probes, and clones corresponding to mRNAswhich are differentially regulated in normal vs. injured nerve wereidentified. This screen resulted in the identification of a 1113 nt cDNAclone which corresponded to an mRNA that was induced in response tonerve injury.

To obtain additional ninjurin cDNA clones, the injured nerve cDNAlibrary was screened with the ³² P-labeled fragment from the initialcDNA clone. Three independent ninjurin clones were isolated. The5'-terminal end of the ninjurin cDNA was obtained using the RACE (rapidamplification of cDNA end) technique using the 5'-AmplifinderRACE kit(Clontech) with RNA prepared from distal sciatic nerve 3 d post-axotomyand oligonucleotides corresponding to nt 117-143 and 177-201 of theninjurin cDNA sequence. The nucleotide sequence of multiple ninjurincDNA clones was determined for both strands using a Model 373 automatedsequencer (Applied Biosystems Inc, Foster City, Calif.).

The nucleotide sequences of these clones and of 5' end RACEamplification products revealed a cDNA that was 1142 nt in length, ingood correspondence with the 1.1 kb size estimated for the ninjurinmRNA. The sequence of this cDNA, which we have named ninjurin, for NerveInjury Induced protein contains an open reading frame of 152 amino acidsthat predicts a 16.3 kD protein. The nucleotide sequence surrounding thepredicted initiator methionine conforms to the Kozak consensus forefficient translation. The 3'-noncoding sequence contains apolyadenylation signal sequence (AATAAA) 22 nucleotides away from the3'-terminal poly(A) tail.

The ninjurin sequence was used to search the non-redundant nucleotideand amino acid sequence databases, but no homology to other proteins wasdetected. Analysis of potential structural motifs of ninjurin usingcomputer programs that predict hydrophobicity (Kyte, et al., (1982) J.Mol. Biol. 157, 105-132), protein localization sites (Nakai et al.,(1992)Genomics 25, 897-911), and protein secondary structure (Rost etal., (1995) Protein Science 4, 521-533), predict two possibletransmembrane domains (residues 72-100, and 118-139). The amino terminalregion of ninjurin, which is predicted to be outside of the cell fromthis analysis, is generally hydrophilic and contains one putativeN-glycosylation site. No signal sequence is present, nor are there othersequence motifs indicative of a specific function or intracellularlocation.

Analysis of RNA expression patterns. Total RNA was prepared from tissuesand samples (10 μg) were electrophoresed on 1% agarose/formaldehyde gelsand blotted onto nylon membranes as previously described (Chomczynski etal., (1987) Anal. Biochem. 162, 156-159). Membranes were probed with a³² p labeled fragment of the ninjurin cDNA. In situ hybridizationhistochemistry was performed using ³² P-labeled antisense or sense RNAprobes transcribed from the ninjurin cDNA (nt 518-1026) on fresh frozentissue samples as previously described (Wanaka et al., (1990) Neuron 5,267-281).

To investigate the expression pattern of ninjurin after sciatic nerveaxotomy, RNA was isolated from the distal segments of the transectednerve as well as the intact, contralateral nerve (control) at selectedtimes after surgery. Ninjurin mRNA levels were very low in uninjuredsciatic nerve, but were dramatically upregulated in the segment distalto the injury (either transection or crush) (data not shown) NinjurinmRNA was detected within 3 hr after injury, and reached peak levels 7-14days after the injury. Ninjurin mRNA levels remained elevated for up to56 days post-transection, whereas after crush injury, which permitsaxonal regeneration, ninjurin expression returned to a low level by day28. This time course correlates well with the nerve regenerationprocess, in which axonal regeneration is generally completed withinseveral months after a crush injury, but is delayed or prevented aftertransection (Daniloff et al., 1986). These changes in ninjurinexpression after nerve injury were confirmed by in situ hybridizationanalysis on nerves (transected vs. intact contralateral) harvested 7days after axotomy. Ninjurin mRNA was detected throughout the distalsegment and at the end of the proximal segment (i.e. proximal neuroma),whereas no signal was observed in the normal sciatic nerve (Data notshown). No difference in intensity was observed throughout the length ofthe distal segment.

To examine the tissue distribution of ninjurin, RNA blot analysis onsamples isolated from a variety of adult rat tissues were performed. Thehighest levels of ninjurin mRNA were found in the liver. In addition,thymus, heart, adrenal gland and spleen also had significant levels ofninjurin transcripts, whereas brain and dorsal root ganglia (DRG) hadlow levels of expression (Data not shown). To examine ninjurinexpression during development, in situ hybridization was performed onrat embryos sacrificed at embryonic days 17 and 19. Ninjurin expressionis observed in a variety of embryonic tissues, with the most abundantexpression observed in tissues where it is highly expressed in the adult(e.g. liver, adrenals, and spleen) (FIG. 3). Tissues hybridized with aninjurin sense probe or tissues treated with ribonuclease prior tohybridization did not give any signal (data not shown). Ninjurin wasalso detected in the vertebra and limbs, where its expression increasedwith increasing embryonic age. The signal was detected primarily inregions of active ossification, such as the terminal areas of the limband vertebral bones. The most intense signal was observed over layers ofdividing chondrocytes, rather than over regions which were alreadyossified. Ninjurin was also highly expressed in the skin, and wasprimarily detected in the epithelium. In the central nervous system,ninjurin expression was very low throughout the embryonic periodexamined.

Generation and analysis of antibodies to ninjurin. A synthetic peptidecontaining an N-terminal cysteine and ninjurin residues 1 through 15 wasconjugated to keyhole limpet hemocyanine using either glutaraldehyde orm-maleimidobenzoyl-n-hydroxysuccinimide ester. The conjugate proteinswere used to immunize rabbits following standard procedures (Cocalico,PA) and anti-ninjurin antibodies were purified by chromatography over anaffinity column in which the peptide was linked to SulfoLink Gel(Pierce) per manufacturer's instructions. Protein blot analysis wasperformed as previously described (Lee et al., (1995) Journal ofBiological Chemistry 270, 9971-9977). Immunohistochemical analysis ofrat tissues was performed on 15 micron sections of 4% paraformaldehydefixed tissues using standard methods. The purified anti-ninjurinantiserum was used at a 1:2500 dilution and specific staining wasdetected with indocarbocyanine (Cy3)-conjugated anti-rabbit IgG (JacksonLab). Glial fibrillary acidic protein (GFAP) was detected using ananti-GFAP monoclonal antibody (BRL) and fluorescein-conjugatedanti-mouse IgG.

These antibodies recognized a 22 kD protein that was present in ratliver and in Chinese hamster ovary (CHO) cells stably transfected with aninjurin expression vector, but not in native CHO cells (Data notshown). The size of the detected protein (22 kD) was larger thanpredicted from the cDNA sequence. Ninjurin was then tested to see if itwas modified post-translationally. Ninjurin was immunoprecipitated withanti-ninjurin antibodies from lysates of the ninjurin-expressing CHOcells, and treated with either peptide:N-glycosidase F to remove anyN-linked carbohydrates, or with alkaline phosphatase to remove anyphosphate groups. After treatment, the proteins were electrophoresed onSDS-polyacrylamide gels. No differences in mobility were observed,suggesting that ninjurin is not modified in either of these manners(data not shown). Furthermore, the mobility of in vitro translatedninjurin also migrates slower than would be expected from its predictedsize.

To further define the ninjurin expression pattern after nerve injury, animmunohistochemical analysis was performed using these anti-ninjurinantibodies. These studies clearly confirmed the upregulation of ninjurinafter sciatic nerve injury. Ninjurin immunoreactive cells were detectedwithin the proximal neuroma, and in the nerve segment distal to the siteof transection (Data not shown). The ninjurin-positive cells werearranged in a parallel linear array, characteristic of the Schwann cellsand their basal lamina (bands of Bungner) that are formed afterdegeneration of the axons and prior to their re-growth. Proximal to thetransection site, Schwann cells did not contain significant amounts ofninjurin, except in the neuroma formed at the transection site whereimmunoreactivity was as intense as in the distal nerve segment. Theidentity of the ninjurin expressing cells as Schwann cells was confirmedby performing immunohistochemistry with antibodies to the Schwann cellmarker S100 and demonstrating a similar pattern of staining (Data notshown). The ninjurin immunoreactivity was blocked by preincubation withthe immunizing peptide, and the pattern of staining was consistent withthe results of our in situ hybridization analysis.

In the central nervous system, cells with a morphology consistent withthat of astrocytes were stained with the anti-ninjurin antibodies (Datanot shown), but no neuronal staining was observed. To confirm theidentity of these cells, simultaneous, dual label immunohistochemistrywas performed using antibodies directed against glial fibrillary acidicprotein (GFAP) and against ninjurin. In this analysis, ninjurin and GFAPimmunoreactivity is present in the same cells, indicating that ninjurinis expressed predominantly by astrocytes.

In agreement with our RNA analysis, an immunohistochemical survey withanti-ninjurin antibodies confirmed that ninjurin is expressed in a widevariety of tissues in the adult rat, including the liver, kidney,thymus, uterus, adrenal gland, and dorsal root ganglia (Data not shown).In the dorsal root ganglia, ninjurin was not detected in neurons but wasexpressed in the satellite cells which ensheath the neuronal cellbodies. In the adrenal gland, ninjurin is expressed throughout thecortex and appears to be on the surface of the cortical cells. Ninjurinimmunoreactivity was also observed on the surface of the hepatocytes ofthe liver. In the kidney, ninjurin was detected in the podocytes and/ormesangial cells of the glomerulus, but other renal cell types werenegative. In the thymus, the thymocytes themselves were negative forninjurin, but the thymic epithelial cells, which provide the propermicroenvironment for lymphocyte maturation, stained intensely with theanti-ninjurin antibodies. Flat cells located on the surface of thethymic cortex and in the area adjacent to the blood vessels alsoexpressed ninjurin. Staining for ninjurin was also observed in theuterus, where the ninjurin-positive cells were found in the myometrium.These cells were distinct from smooth muscle cells, and theirdistribution and morphology was consistent with fibroblasts of theassociated connective tissues. Taken together, these results demonstratethat ninjurin immunoreactive cells are found in a wide variety oftissues.

Cell culture and transfection. Chinese hamster ovary (CHO) cells werecultured in F-12 medium supplemented with 10% fetal calf serum (FCS). Tcell leukemia cells (Jurkat) were grown in RPMI1640 with 10% FCS. Theexpression vector pCMV-ninjurin was constructed by inserting theninjurin cDNA into the plasmid pCMVneo (Brewer, C.B. (1994). Methods inCell Biol. 43, 233-245). The pCMVneo or pCMV-ninjurin plasmids wastransfected into CHO cells via calcium phosphate precipitation and intoJurkat cells via electroporation. Stable transfectants were selected bygrowth in medium containing G418 (400 μg/ml) and individual clones wereisolated by limiting dilution. Primary cultures of sympathetic neuronsfrom superior cervical ganglia (SCG) were prepared by dissecting tissuefrom E20-21 rat embryos as previously described (Martin et al., (1988)J. Cell. Biol. 106, 829-844). Primary cultures of neurons from dorsalroot ganglia (DRG) were prepared from E17 rat embryos as previouslydescribed (Eichler et al., (1989) Brain Res 482, 340-346). Dissociatedneurons were plated on confluent monolayers of either control CHO cellsor CHO cell expressing ninjurin in 24-well plates. Cultures were grownin a medium which consists of 90% Eagles minimal essential medium(Gibco), 10% FCS, 20 μg/ml each of fluoro-deoxyuridine and uridine, and50 ng/ml mouse NGF (gift from E. Johnson, Washington U.). Six hr afterplating, the cells were fixed with 4% paraformaldehyde in PBS, andimmunohistochemistry using anti-neurofilament H antibodies wasperformed. Following previously established criteria (Gennarini et al.,(1991) Neuron 6, 595-606), approximately 50 neurites were chosen formeasurement which each emerged from a single distinguishable cell body,were longer than the diameter of the neuronal cell body, and wereidentifiable over their entire length. The length of each neurite wasmeasured after tracing them from the photomicrographs onto grided (2 mm)transparency film. Statistical analysis was performed using Sigma Plot(version 3.0).

Surface labeling of cells and immunoprecipitation of ninjurin. Cellsurface proteins of the ninjurin transfected CHO cells wereradioiodinated with Na¹²⁵ I (Amersham) using water-soluble Bolton-Hunterreagent (Pierce) as per manufacturer's instructions. Immunoprecipitationof ninjurin from these lysates was performed using anti-ninjurinantibodies as described previously (Fahrner et al., (1990) Mol. Cell.Biol. 10, 6454-6459). Immunoprecipitations were also performed using amonoclonal antibody directed against the cytoplasmic proteinphospholipase Cy I monoclonal antibody (Upstate Biotechnology, Inc.) asa control. ³⁵ S-labeled phospholipase Cγ1 was immunoprecipitated fromninjurin transfected CHO cells grown in medium containing [³⁵S]methionine (120 mCi/ml final concentration, Amersham) for 16 hr.

The pattern of ninjurin immunoreactivity observed in theimmunohistochemical analysis suggested that ninjurin was localized tothe cell surface. This was most apparent in the adrenal gland and liver,where ninjurin immunoreactivity was clearly restricted to the adrenalcortical and hepatocyte cell surfaces, suggesting that ninjurin iseither a membrane protein or possibly a secreted factor which adsorbs tothe cell surface or extracellular matrix (data not shown). To furtherexamine ninjurin localization, we attempted to immunoprecipitate it fromcell lysate vs. medium conditioned by CHO cells expressing ninjurin.This experiment demonstrated that ninjurin was only present in the celllysate (data not shown), suggesting that ninjurin is likely to be amembrane protein. To directly demonstrate that a portion of the ninjurinmolecule is located outside of the cell, we labeled ninjurin expressingCHO cells with ¹²⁵ I using the Bolton-Hunter reagent under conditionswhich selectively label proteins on the cell surface (Thompson et al.,(1987) Biochem. 26, 743-750). After labeling the cells,immunoprecipitations were performed with anti-ninjurin specificantibodies as well as with antibodies directed against the intracellularprotein phospholipase Cγ1, and the immunoprecipitated proteins wereseparated by gel electrophoresis and visualized by autoradiography (Datanot shown). It is clear that the anti-ninjurin antibodies precipitatedan intensely labeled 22 kD protein, whereas no signal was detected withthe anti-phospholipase Cγ1 antibody. Phospholipase Cγ1 was effectivelyprecipitated with this antibody using ³⁵ S-methionine-labeled cells(lane 5), indicating that are ¹²⁵ I labeling protocol did not labelintracellular proteins. These results therefore strongly suggested thatninjurin is a plasma membrane protein, as predicted from sequenceanalysis.

Cell adhesion assays. The cell surface localization of ninjurin combinedwith its expression in glia after nerve injury and its expression inepithelial cells in a number of tissues suggested that it might beinvolved in cellular adhesion. Aggregation assays were performed usingJurkat cells (Shimizu et al., (1990) Nature 345, 250-253) stablytransfected with pCMV-ninjurin or pCMVneo (nonrecombinant control). Thecells were washed twice with RPMI1640 containing 5 mM Hepes buffer,suspended to a concentration of 2×10⁶ cells/ml in RPMI1640 with 10% FCS,and 100 μl of the cell suspension were added to each well of aflat-bottomed 96-well microtiter plate. The formation of aggregates wasexamined after 1 hr using phase contrast microscopy. For demonstratinghomophilic binding, aggregation experiments were performed using amixture of ninjurin stable transfectants and non-transfected cells. Theninjurin positive cells were stained red with 1 mM CMTMR(5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) andcontrol cells were stained green with 1 mM CMFTA(5-chloromethylfluoresceindiacetate) as described in the manufacturer'sprotocol (Molecular Probes). The cells were resuspended to 1×10⁶cells/ml, and 3 ml of the mixed cell suspension was allowed to formaggregates in 6-well culture plates. The composition of the aggregateswas monitored by fluorescence microscopy.

Protein blot analysis was used to demonstrate that ninjurin is expressedin cells transfected with CMV-ninjurin, but is absent or present at verylow levels in native Jurkat cells (data not shown). Incubation ofninjurin-expressing cells resulted in the formation of large aggregatesthat were readily demonstrable after 1 hr, whereas the control Jurkatcells formed only a few small aggregates (Data not shown). Aggregateformation depended on the presence of either Mg, Mn or Ca ions; noaggregates were detected when these assays were performed in thepresence of 1 mM EDTA, suggesting that ninjurin mediated adhesion isdependent on divalent cations (data not shown).

For aggregation experiments using CHO cells, cells (ninjurin transfectedand control) were harvested from confluent plates by treatment withtrypsin, washed with F-12 medium, and resuspended at 1×10⁶ cells/ml inF-12 with 10% FCS. Aggregation assays were performed on a rotary shakerat 80 rpm. Quantitation of aggregate formation was monitored at 15 minintervals by counting the number of particles using a hemocytometer(Takeichi, M. (1977). J. Cell. Biol. 75, 464-474). The extent of theaggregate formation is inversely proportional to the number ofparticles, and was determined by calculating an index N₁ /N₀, where N₁and N₀ were the total particle numbers at incubation times t and 0,respectively (Takeichi, 1977). All of the aggregation experiments wereperformed at 37° C.

A decrease in the number of particles with time was observed with thetransfected cells, indicating the formation of larger aggregates withninjurin-expressing cells (data not shown) (Takeichi, 1977). Theseresults provide further support that ninjurin is involved in cellularadhesion as its effects are observed in multiple cell types.

To determine whether the adhesion mediated by ninjurin could occur via ahomophilic mechanism, aggregation experiments on mixtures of transfectedand non-transfected Jurkat cells that were differentially stained withfluorescent dyes were performed. Equal amounts of ninjurin-positivecells (colored red) and control cells (colored green) were mixedtogether and the resulting aggregates were examined by fluorescencemicroscopy. The aggregates consisted predominantly of red,ninjurin-positive cells, with some non-transfected cells adhering to thesurface (Data not shown). None of the aggregates were composed primarilyof non-transfected cells, nor wer aggregates with a mosaic pattern ofthe two cell types observed.

The demonstration that ninjurin can mediate adhesion via homophilicbinding, encouraged us to investigate its expression on neurons, as itmay play an important role in nerve regeneration. We therefore examinedcultures of sympathetic neurons dissected from the superior cervicalganglia (SCG) of E21 rat pups. After culturing the neurons for 5 days inNGF and the anti-mitotic bromodexoyuridine (to remove non-neuronalcells), immunohistochemical analysis was performed using eitheranti-ninjurin or anti-neurofilament H antibodies (Data not shown). Thepattern of staining for these two molecules is very similar, withintense immunoreactivity present in the cell bodies and extending outinto the axonal projections. The co-localization of ninjurin withneurofilament H, along with the detection of ninjurin immunoreactivityin the neuronal projections, indicates that ninjurin is present inlocations where ninjurin homophilic binding could occur between Schwanncells and neurons.

Ninjurin is expressed by DRG neurons after injury and is transported tothe site of injury. To promote nerve regeneration via homophilicbinding, ninjurin should be expressed by neurons as well as Schwanncells after nerve injury. To examine this possibility, ninjurinexpression was examined in the dorsal root ganglion (DRG) after sciaticnerve transection.

Immunohistochemistry with anti-ninjurin antibodies revealed thatninjurin was detectable 1 day after nerve transection in the DRGneuronal cell bodies (Data not shown). Ninjurin expression was notdetected in DRG neurons from the contralateral uninjured side,suggesting that neuronal synthesis of ninjurin is induced by axotomy. Wenext tested whether ninjurin was anterogradely transported down theaxon. For this purpose, the sciatic nerve was ligated and 5 to 8 mmdistal to the ligation site the nerve was transected. As a control,sciatic nerves were ligated but no axotomy was performed. After 1 day,the nerves were harvested and ninjurin was detected usingimmunohistochemistry. Ninjurin staining was intense proximal to theligation site in the nerve that had been transected (Data not shown),suggesting that ninjurin had been transported down the axon. Incontrast, only a minor amount of ninjurin staining was observed aroundthe ligation site in the non-transected nerve, presumably representing alocal response to the ligation itself. Taken together, these resultsindicate that ninjurin is upregulated in neurons after nerve injury andis subsequently anterogradely transported to the site of injury, whereninjurin is also expressed by Schwann cells.

Ninjurin promotes neurite outgrowth from DRG neurons cultured in vitro.

To test directly whether ninjurin can promote neurite outgrowth, weexamined DRG neuron/CHO cell co-cultures as previously used to analyzepromotion of neurite outgrowth by neuronal F3 glycoprotein (Gennarini etal., 1991) and NCAM (Doherty et al., (1989) J. Cell Biol. 109, 789-798).DRGs from E17 rat embryos were dissected, dissociated and plated at lowdensity onto confluent monolayers of either CHO cells expressingninjurin or control CHO cells. Six hr after plating, the cells werefixed and stained with antibodies to neurofilament H to visualize theneurites. Representative photomicrographs of these cultures demonstratean increase in neurite outgrowth from neurons cultured on CHO cellsexpressing ninjurin (Data not shown). To quantitate this effect,approximately 50 neurons were randomly selected from each growthcondition, and the length of the longest neurite per neuron, which didnot have contacts with nearby neurons, was measured. Neurons grown for 6hr on CHO cells expressing ninjurin had neurites with an average lengthof 569±134 μm, whereas neurites from neurons grown on control CHO cellswere 297±104 μm in length. These results indicate that ninjurin promotesan increase in the extent of neurite outgrowth.

Example 2 Characterization of the binding domain of ninjurin

To directly investigate whether the N-terminal extracellular hydrophilicregion is responsible for the observed homophilic adhesion, partiallyoverlapping synthetic peptides whose sequences collectively encompassthe entire predicted ninjurin extracellular domain were tested for theirability to inhibit the ninjurin- mediated adhesion. Aggregation assayswith the ninjurin-expressing Jurkat cell transfectants were performed,and the degree of aggregation was examined after 60 minutes by measuringthe number of cells incorporated in aggregates. Peptide 2 inhibitedaggregation at a concentration of 0.1 mg/ml, and aggregation wascompletely abolished at concentrations above 1 mg/ml. In constrast,peptides 1 and 4 had no effect on aggregation at either of theseconcentrations. These results indicate that the ninjurin amino terminusis indeed located extracellularly, and that residues 16 to 45 (sequencewhich corresponds to peptide 2) contains a domain that is critical forbinding.

To further delimit the site of interaction, an additional 4 peptides(peptides 5 through 8) were tested for their ability to inhibitninjurin-mediated aggregation (Data not shown). A peptide correspondingto ninjurin residues 26 to 37 showed inhibitory activity comparable topeptides 2 or 3. Inhibition of aggregation was observed atconcentrations higher than 0.4 mg/ml and was completely abolished atconcentrations above 2 mg/ml. To identify the amino acids most criticalfor ninjurin-mediated adhesion, peptides (9-15) stretching from residue26 to 37, each containing single amino acid mutations, were tested inthe aggregation assay. The ability of each of these peptides to inhibitninjurin-mediated adhesion in a dose-dependent manner was examined. Theseven residues between prolines 27 and 35 (P27 and P35) were mostimportant. Mutation of the Arg or Trp residues resulted in dramaticdecreases in the ability to inhibit aggregation, implying that these 4residues play an important role in ninjurin-ninjurin molecular contact.Mutations made to the non-charged residues including Gly-30, Leu-31 andAsn-33 did not alter the ability to inhibit aggregation, indicating thatthese residues are not critical to the interaction. Curiously, one ofthe mutations (Asn-33 to Leu33) resulted in a peptide with greaterinhibitory activity than wild type, suggesting that it interacts withninjurin very strongly.

The ninjurin adhesion motif contains a tryptophan and a cluster ofarginine residues. None of the previously reported homophilic adhesionmolecules contains this peptide motif, or a combination of tryptophanand arginines as functionally relevant residues. The peptide inhibitionexperiments showed that the replacement of tryptophan by alaninedramatically decreased the peptide's inhibitory effect, suggesting thatthis tryptophan residue plays a major role in ninjurin-ninjurininteractions. This tryptophan residue may be directly involved in thephysical interaction between ninjurin molecules, or, perhaps, it isnecessary for the overall structure of the domain. Replacement of thearginines showed significant but less dramatic effects on ninjurinadhesion. Although these arginines appear to be essential, directinteractions between these positively charged residues on opposingmolecules seems unlikely. Perhaps these residues contribute to ninjurinadhesion indirectly by neutralizing negatively charged regions elsewherewithin the molecule. Interestingly, even though ninjurin binding isdependent on divalent cations, the ninjurin adhesion motif does notcontain acidic residues commonly associated with cation binding motifs.Overall ninjurin contains 13 Glu and Asp residues, with 6 of theselocated within the amino terminal 23 residues. Perhaps this acidicregion is related to ninjurin's cation dependence and thus plays a rolein the formation of the functional ninjurin adhesion domain.

The demonstration that the inhibitory peptide we identified via cellaggregation assays was able to reverse the ninjurin-stimulated neuriteoutgrowth from neurons indicates that the biological function ofninjurin is related to its adhesive properties. In addition, the basaladhesion observed for wild type Jurkat cells was abolished in thepresence of peptides containing ninjurin's adhesion motif (data notshown). This suggests that other molecule(s) expressed by Jurkat cellspossibly share the ninjurin adhesion motif. If other molecules with theninjurin-like adhesion motif exist, then the possibility that ninjurinmay participate in heterophilic interactions as well homophilicinteractions must be considered. This would greatly extend the number ofinteractions, and potentially the number of functions, in which ninjurinis involved.

Our expression analysis revealed that ninjurin is present in a widevariety of tissues (e.g. thymus, kidney, liver, adrenal gland) inaddition to the nervous system and is predominantly expressed inepithelial cells, suggesting ninjurin is important in the developmentand/or function of a number of tissues. For instance, its presence inthe thymus may indicate a role in thymocyte development, as adhesionmolecules on thymic epithelial cells are thought to be important in thisprocess (Patel et al., (1993) Immunol 5, 283-292). The isolation ofninjurin and its identification as a new adhesion protein that isexpressed in a number of tissues during embryogenesis as well asadulthood suggest that it may play an important role in the properdevelopment and function of a variety of tissues, in addition to itsrole in nerve regeneration.

Ninjurin mutants fail to support aggregation. Aggregation inhibitionexperiments using synthetic peptides revealed that residues 26 to 37(the ninjurin adhesion motif) are responsible for ninjurin-mediatedhomophilic cellular adhesion. However, these current results do notdistinguish whether homophilic interactions result from (1) a directinteraction between this motif on two ninjurin molecules or (2) aninteraction with this motif and another region on the second ninjurinmolecule. To investigate these possibilities, Jurkat cell linesexpressing ninjurin molecules containing a double mutation (Arg28 to Asnand Asn29 to Ala) in the ninjurin adhesion motif was generated. Proteinblot and immunohistochemical analyses of these cell lines confirmed thatthe expression levels for wild type and mutant ninjurin molecules werecomparable, and that the ninjurin mutants were expressed on the cellsurface (Data not shown). Assays with these ninjurin transfected celllines revealed that Jurkat cells stably expressing either of theninjurin mutants showed no increase in aggregation over native Jurkatcells (Data not shown). These results demonstrate that the motifidentified using peptides and aggregation inhibition is clearlynecessary for ninjurin-mediated adhesion. To determine whether themutant molecules could interact with wild type ninjurin, we performedaggregation assays on mixtures of cells expressing either wild type ormutant ninjurin molecules. When equal amounts of the two types of cellswere mixed together, the aggregates contained predominantly wild typeninjurin-expressing cells (Data not shown). A mosaic pattern, containingequivalent amounts of the two types of cells, was not observed. Takentogether, these results indicate that interactions between the ninjurinadhesion motif (residues 26 to 37) on both ninjurin molecules areresponsible for the homophilic adhesion mediated by ninjurin.

Ninjurin-stimulated neurite outgrowth is mediated through the adhesionmotif. To determine whether the ninjurin promotion of neurite extensionfrom primary DRG neurons occurs via ninjurin's adhesive properties, thepeptides were tested for the ability to inhibit neurite outgrowth. DRGneurons from E17 rat embryos were dissociated and seeded onto confluentmonolayers of CHO cells, either native or expressing ninjurin. Theneuronal cultures were treated with peptides (0.5 mg/ml) correspondingto either the native or mutant sequences of the ninjurin adhesion motif,and neurite extension was monitored. As expected, neurites from neuronsgrown on CHO cells expressing ninjurin showed increased neuriteoutgrowth compared to those grown on native CHO cells. However, theneurite outgrowth promoting effect of ninjurin was inhibited whenneurons were treated with the peptide of native sequence, whereas thepeptide containing a mutation in this sequence (PPRWGLRNRPIN) (SEQ IDNO: 21) had no significant inhibitory effect on neurite extension by DRGneurons. These results indicate that the adhesion motif is required forninjurin-stimulated neurite outgrowth, suggesting that the adhesiveproperties of ninjurin play an important functional role in thisprocess.

Example 3 Cloning of human ninjurin proteins

Rat ninjurin nucleic acid sequence was used to search for homologoussequence in the EST database by basic local alignment search tool(BLAST: Altschul, S. F., et al. J. Mol. Biol. 215:403-410, 1990). HumanEST clones yd37d12(T84142, T89382) and yu87c07(H91351, H91056),yf8b06(R06312, R06257), yd98c05 (T89765, T89491) were obtained. Thenucleotide sequences of the entire clones were determined . Allnucleotide sequencing was performed using fluorescent dye terminatortechnology per manufacturer's instructions on an Applied Biosystemsautomated sequencer model#373 (Applied Biosytstems, Foster City,Calif.). Plasmid DNA for sequencing was prepared by using WizardMiniprep kit (Promega Corp., Madison, Wis.) according to themanufacturer's instructions. To obtain 5' end of each of the clones,oligonucleotide primers corresponding to the reverse complementarysequence of the human EST clones (yd37d12, and yu87c07) were used forthe rapid amplification of the cDNA ends (RACE) technique (Frohman, M.A.Methods in Enzymology 218:340-356, 1993) using the Human Brain (forninjurin1-variant, ninjurin2) and Human Adrenal (for ninjurn1) MarathonRACE kit (CLONTECH, Palo Alto, Calif.) per manufacturer's instructionswith minor modifications. Briefly, using nested reverse primers forninjurinl (#40365: 5'-

AGGGCAGGCAGCATCCAGGGTCCT (SEQ ID NO: 28), and #40373:5'-TGTCCGGGTTGTTAAGGTCGTACTT) (SEQ ID NO: 29) and ninjurin2(#30741:5'-GAAGACCAAGATGGTGGCTGCGTTG (SEQ ID NO: 30) and#10348:5'-TTCAGGTTCAGCCGTGCAATGACCA) (SEQ ID NO: 31 ) in combinationwith the primers to the adaptor at cDNA ends (AP1, AP2; provided in thekit), the 5' end of the ninjurin 1 and ninjurin 2 were amplified by twosuccessive PCR reactions using the following parameters. For ninjurin1:using human adrenal cDNA as a template, 1st reaction: #40365 and AP1,94C. for 2 min, 68 C. for 30 sec and 68C. 2 min, followed by decreasinganealing temperature by 0.5 C. per each cycle for 16 cycles, then 94 C.2 min, 60 C. 30 sec, and 68 C. for 2 min for 20 cycles. 2nd reaction:#40373 and AP2, 94 C. for 2 min, 68 C. for 2 min 30 sec for 25 cycles.For ninjurin 2, human brain cDNA was used as a template with 1streaction:#30741 and AP1, 94 C. for 30 sec, 68 C. for 30 sec, and 68 C.for 2 min, followed by decreasing anealing temperature by 0.5 C. pereach cycle for 19 cycles, then 94 C. 2 min, 58 C. 30 sec and 68 C. for 2min for 20 cycles. 2nd reaction: #10348 and AP2: 94 C. for 30 sec, 72 C.for 2 min for 5 cycles, 94 C. for 30 sec, 70 C. for 2 min for 5 cycles,and 94 C. for 30 sec, 68 C. for 2 min for 25 cycles.) The RACE-PCRproducts after secondary PCR were cloned into pBluescript KS+ andsequenced. The resulting sequence of ninjurin 2 was different from thesequence of the clone yu87c07 at its 5' end, and the RACE-PCR productwas termed ninjurin2 and the clone yu87c07 was termed variant 2 ofninjurin 2. The nucleotide sequence of the clone yd98c05 was identicalto the sequence of ninjurin1 only in its 3' end region, and termed asninjurin1-variant. The nucleotide sequence of the clone yf08b06 wasidentical to the sequence of ninjurin 2 only in its 3' end region, andtermed as ninjurin2-variant 1. Nested reverse primers specific to theninjurin 1-variant (#30749: 5'-TCAGTCGCATTTAGGATGAGTTTTG (SEQ ID NO:32), and #10391: 5'-GGATGAGTTTTGTCGCCCAGAGAA) (SEQ ID NO: 33) weresynthesized and used for RACE-PCR to amplify 5' end of theninjurinl-variant cDNA (1st PCR: #30749 and AP1, 94 C. for 30 sec, 68 C.for 30 sec, and 68 C. for 2 min, followed by decreasing anealingtemperature by 0.5 C. per each cycle for 19 cycles, then 94 C. 2 min, 58C. 30 sec and 68 C. for 2 min for 20 cycles, 2nd PCR: #10391 and AP2, 94C. for 30 sec, 72 C. for 2 min for 5 cycles, 94 C. for 30 sec, 70 C. for2 min for 5 cycles, and 94 C. for 30 sec, 68 C. for 2 min for 25cycles). The RACE-PCR product after secondary PCR was cloned intopBluescript KS+ and sequenced.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 34                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Asp Xaa Asn Asp Asn                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Lys Tyr Ser Phe Asn Tyr Asp Gly Ser Glu                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1142 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CTGGGCGGCC CGGGCGGCCG CACCATGGAT CCCGGCACCG AGGAGTACGA GC -            #TCAACGGC     60                                                                 - - GACCTGCGCC CTGGCTCTCC CGGTTCCCCC GACGCCTCGC CACCCCGCTG GG -            #GTTTGAGG    120                                                                 - - AACCGGCCCA TCAATGTAAA CCATTACGCC AACAAGAAGA GCGCCGCGGA GA -            #GCATGCTG    180                                                                 - - GACATCGCAC TGCTCATGGC CAACGCGTCG CAGCTCAAGG CCGTGGTGGA GC -            #AGGGCAAT    240                                                                 - - GAGTTCGCCT TCTTCGTGCC CCTCGTGGTG CTCATCTCCA TCTCTCTCGT GC -            #TGCAGATC    300                                                                 - - GGAGTGGGCG TGCTGCTCAT CTTCCTGGTC AAGTATGACC TCAACAACCC AG -            #CCAAGCAC    360                                                                 - - GCCAAGCTGG ACTTCCTTAA CAACCTGGCC ACGGGGCTGG TTTTCATCAT CG -            #TGGTGGTC    420                                                                 - - AACATCTTCA TTACGGCCTT TGGAGTCCAG AAGCCTGTCA TGGACGTGGC AC -            #CCCGGCAG    480                                                                 - - TAGGATGCCC AGAGACCTTG AAGGTATCTG ACCTGCAGCC CAGCTGTCCA GA -            #CCCCTGCA    540                                                                 - - ACTGCTGTAT CCCCAAGGCA TCCCTCTCCT GTTCACAGCC CAAGGTGGCC TC -            #CGCTGGAC    600                                                                 - - CATGGTCAAG GATGGACTTC CGTCCACCTG TGACTGCTGC GTGGGCGGCC AC -            #CCGAGGCG    660                                                                 - - TGTGGGAACT GGATGCAAAG CCATGAAGAT CAGAACTGGA CAGTTCCACC GA -            #AACCCACG    720                                                                 - - CCCAGAGGAT GATCACTGCC CGCCCAAGGA CATGCAGGAA ATCCATGATT GG -            #ACTCGATG    780                                                                 - - AGGGGCCAGA ACTGATCTCT GTCTCAGGAC ATTCCAGAAG GACCAGGATA TG -            #CCCCTCCC    840                                                                 - - TTTGCTGATA CACCAGTGAC CCTACTTCTC ATGGAGCATG CACAGGTCAC CC -            #TGGAGACT    900                                                                 - - GCTCCCTTTG TTGTTTCCTG ACCCAGGGAC CTTGGACAGT CATCAGTACC TG -            #CTGGCTCC    960                                                                 - - AGCCTCAGTG CCTGGGCTTG GCAGTGTCTC TTGGCATCGA GAGGCAGCCA TG -            #CCTGTGGG   1020                                                                 - - GGCTGCAGGT CATCCTGGTA CCTTCTACCA GTAGTGACTT GGGAAGAGCC CC -            #ACCCCCCA   1080                                                                 - - ACCCAGGGGC TCAGGCCCCA ATTTTCTAAT CAGGAATGAC AATAAAGCTT AT -            #GTCTTCCC   1140                                                                 - - CC                  - #                  - #                  - #                1142                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Asp Pro Gly Thr Glu Glu Tyr Glu Leu As - #n Gly Asp Leu Arg Pro      1               5   - #                10  - #                15               - - Gly Ser Pro Gly Ser Pro Asp Ala Ser Pro Pr - #o Arg Trp Gly Leu Arg                  20      - #            25      - #            30                   - - Asn Arg Pro Ile Asn Val Asn His Tyr Ala As - #n Lys Lys Ser Ala Ala              35          - #        40          - #        45                       - - Glu Ser Met Leu Asp Ile Ala Leu Leu Met Al - #a Asn Ala Ser Gln Leu          50              - #    55              - #    60                           - - Lys Ala Val Val Glu Gln Gly Asn Glu Phe Al - #a Phe Phe Val Pro Leu      65                  - #70                  - #75                  - #80        - - Val Val Leu Ile Ser Ile Ser Leu Val Leu Gl - #n Ile Gly Val Gly Val                      85  - #                90  - #                95               - - Leu Leu Ile Phe Leu Val Lys Tyr Asp Leu As - #n Asn Pro Ala Lys His                  100      - #           105      - #           110                  - - Ala Lys Leu Asp Phe Leu Asn Asn Leu Ala Th - #r Gly Leu Val Phe Ile              115          - #       120          - #       125                      - - Ile Val Val Val Asn Ile Phe Ile Thr Ala Ph - #e Gly Val Gln Lys Pro          130              - #   135              - #   140                          - - Val Met Asp Val Ala Pro Arg Gln                                          145                 1 - #50                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1235 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GCGGCCTGGG CGGCCGCACC ATGGACTCGG GAACCGAGGA GTACGAGCTC AA -             #CGGCGGCC     60                                                                 - - TGCCTCCGGG CACACCCGGC TCCCCGGACG CCTCGCCGGC CCGCTGGGGC TG -            #GAGGCACG    120                                                                 - - GGCCCATCAA CGTGAACCAT TACGCCAGCA AGAAGAGCGC AGCCGAGAGC AT -            #GCTGGACA    180                                                                 - - TCGCGCTGCT GATGGCCAAC GCGTCCCAGC TGAAGGCCGT CGTGGAACAG GG -            #CCCCAGCT    240                                                                 - - TCGCCTTCTA TGTGCCCCTG GTGGTCCTCA TCTCCATCTC CCTTGTGCTG CA -            #GATCGGCG    300                                                                 - - TGGGGGTGCT GCTCATCTTC CTTGTCAAGT ACGACCTTAA CAACCCGGAC AA -            #GCACGCCA    360                                                                 - - AGCTGGACTT CCTCAACAAC CTGGCCACGG GCCTGGTGTT CATCATCGTG GT -            #AGTCAACA    420                                                                 - - TCTTCATCAC GGCCTTCGGG GTCCAGAAGC CCTTGATGGA CATGGCACCC CA -            #GCAGTAGG    480                                                                 - - ACACCCAGGA CCCTGGATGC TGCCTGCCCT GCAACTCAGC TGCCCGACCC CA -            #GGAGTCGC    540                                                                 - - CATACCTGTG AGGTGTCCAC CTCCCTGCAC ATGGCACTAC CCAGACTGCC AG -            #AGCCCAGG    600                                                                 - - CTGGCCTCAT CTGCACCATG TCCCCGGACC AGCCCTTGCT CTGACTGCGG CC -            #AAGCACCA    660                                                                 - - CGCAGGAGGC CACTCTTGTC TCTCASCAGC TGTTCCCAGG AGGCAGCTCC CT -            #CCTGGCAC    720                                                                 - - ATGGGGGCTG GCACAATAGC CCAGAGGGTC AGAACTGGAC AGCTGCAGAG AC -            #CTGTGCCC    780                                                                 - - AGAGAAGGGT CTCGACCCAC TCAAGGACAC ACAGCAGGTC CGTGGATGGG CT -            #GGATGAGT    840                                                                 - - GACCAGGGCC AGCCTCTGTC TCAGGACATT CCAGAAGGAC AAGGAGATGT CT -            #CTCCCTCT    900                                                                 - - CCCAAAGCAC CAGCGTCCCT GCCTCCCGTG GGCCCTGTCC GGGTTGCCCC TG -            #GTGACCCC    960                                                                 - - AGCCTCTGTC CACTTCCTAA CCCAGGGACC CTGCACAGCC AGAACTGCCT TT -            #GGCCCTAC   1020                                                                 - - GGATGGCCAC TGGCTCTGGT CTAAAGTGCC TGGGCTTGGT GGCCATCAAG AG -            #GGAGCCAG   1080                                                                 - - TCAGGCCTGT GAGGGCCGTA GACCTTGTAT ATACCCTGCA CCAGCAGTGA CC -            #GGGCAGAG   1140                                                                 - - CCCAACCCCC TCCACGGGGG TCCCAGCACC CACTTTTCTA ATCATGAATG AA -            #CAATAAAG   1200                                                                 - - CCCACGCTCT TTGTCAGGCA AAAAAAAAAA AAAAA       - #                       - #     1235                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Asp Ser Gly Thr Glu Glu Tyr Glu Leu As - #n Gly Gly Leu Pro        Pro                                                                             1               5   - #                10  - #                15              - - Gly Thr Pro Gly Ser Pro Asp Ala Ser Pro Al - #a Arg Trp Gly Trp Arg                  20      - #            25      - #            30                   - - His Gly Pro Ile Asn Val Asn His Tyr Ala Se - #r Lys Lys Ser Ala Ala              35          - #        40          - #        45                       - - Glu Ser Met Leu Asp Ile Ala Leu Leu Met Al - #a Asn Ala Ser Gln Leu          50              - #    55              - #    60                           - - Lys Ala Val Val Glu Gln Gly Pro Ser Phe Al - #a Phe Tyr Val Pro Leu      65                  - #70                  - #75                  - #80        - - Val Val Leu Ile Ser Ile Ser Leu Val Leu Gl - #n Ile Gly Val Gly Val                      85  - #                90  - #                95               - - Leu Leu Ile Phe Leu Val Lys Tyr Asp Leu As - #n Asn Pro Asp Lys His                  100      - #           105      - #           110                  - - Ala Lys Leu Asp Phe Leu Asn Asn Leu Ala Th - #r Gly Leu Val Phe Ile              115          - #       120          - #       125                      - - Ile Val Val Val Asn Ile Phe Ile Thr Ala Ph - #e Gly Val Gln Lys Pro          130              - #   135              - #   140                          - - Leu Met Asp Met Ala Pro Gln Gln                                          145                 1 - #50                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 907 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CCCACGCAGT CTGTTCCCGG CACCCGGTGC GTGTGAAGGG ACTTGAGGGC AG -             #CGAGATGG     60                                                                 - - AATCAGCAAG AGAAAACATC GACCTTCAAC CTGGAAGCTC CGACCCCAGG AG -            #CCAGCCCA    120                                                                 - - TCAACCTGAA CCATTACGCC ACCAAGAAGA GCGTGGCGGA GAGCATGCTG GA -            #CGTGGCCC    180                                                                 - - TGTTCATGTC CAACGCCATG CGGCTGAAGG CGGTGCTGGA GCAGGGACCA TC -            #CTCTCATT    240                                                                 - - ACTACACCAC CCTGGTCACC CTCATCAGCC TCTCTCTGCT CCTGCAGGTG GT -            #CATCGGTG    300                                                                 - - TCCTGCTCGT GGTCATTGCA CGGCTGAACC TGAATGAGGT AGAAAAGCAG TG -            #GCGACTCA    360                                                                 - - ACCAGCTCAA CAACGCAGCC ACCATCTTGG TCTTCTTCAC TGTGGTCATC AA -            #TGTTTTCA    420                                                                 - - TTACAGCCTT CGGGGCACAT AAAACAGGGT TCCTGGCTGC CAGGGCCTCA AG -            #GAATCCTC    480                                                                 - - TCTGAATGCA GCCTGGGACC CAGGTTCTGG GCCTGGAACT TCTGCCTCCT TC -            #CTCCGTGA    540                                                                 - - TCTGCCAGGC TCGTGGGCAC TTTCCACAGC CCAGGAGAGC TTCTGAAAGG AC -            #AGTATAGC    600                                                                 - - TGCCCTTGCT CCCTACCCAC AGCACCTGAG TTAAAAAGTG ATTTTTATGT TA -            #TTGGTCTA    660                                                                 - - AGGGACTTCC ATCTTGGTCT GAAGTCCTGA GCTCAGACGC AGGTACTGCC AG -            #CCATACCT    720                                                                 - - TCCTGGTAGC ATCTGCTGGA CCTAAGTAAG GCATGTCTGT CTAAGGCCAA GT -            #CTGCCCGG    780                                                                 - - CTTAAGGATG CTGGTTCTGA CTCTACCCCA CTGCTTCCTT CTGCTCCAGG CC -            #TCAATTTT    840                                                                 - - CCCTTCTTGT AAAATGGAAT CTATATCTAT AAAGGTTTCT TCAAATCCAA AA -            #AAAAAAAA    900                                                                 - - AAAAAAA                 - #                  - #                       - #         907                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 142 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Glu Ser Ala Arg Glu Asn Ile Asp Leu Gl - #n Pro Gly Ser Ser        Asp                                                                             1               5   - #                10  - #                15              - - Pro Arg Ser Gln Pro Ile Asn Leu Asn His Ty - #r Ala Thr Lys Lys Ser                  20      - #            25      - #            30                   - - Val Ala Glu Ser Met Leu Asp Val Ala Leu Ph - #e Met Ser Asn Ala Met              35          - #        40          - #        45                       - - Arg Leu Lys Ala Val Leu Glu Gln Gly Pro Se - #r Ser His Tyr Tyr Thr          50              - #    55              - #    60                           - - Thr Leu Val Thr Leu Ile Ser Leu Ser Leu Le - #u Leu Gln Val Val Ile      65                  - #70                  - #75                  - #80        - - Gly Val Leu Leu Val Val Ile Ala Arg Leu As - #n Leu Asn Glu Val Glu                      85  - #                90  - #                95               - - Lys Gln Trp Arg Leu Asn Gln Leu Asn Asn Al - #a Ala Thr Ile Leu Val                  100      - #           105      - #           110                  - - Phe Phe Thr Val Val Ile Asn Val Phe Ile Th - #r Ala Phe Gly Ala His              115          - #       120          - #       125                      - - Lys Thr Gly Phe Leu Ala Ala Arg Ala Ser Ar - #g Asn Pro Leu                  130              - #   135              - #   140                          - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1374 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CCACCCGGGC AGGTCCACGC TCAGCCTTGT TTTGTTTTGT TTTGTTTTGT TT -             #TTGAAACC     60                                                                 - - GGGTCTTGCT GTGTCTTGAT CACAGCTCAC TGCAGTCTCA ACCTCCCAGG CT -            #CAAGCGAG    120                                                                 - - CCTTCTGCCT CAGCCTCCCA GGTAGGCTGG ACCACAGCTA TATGCCATCA TG -            #CCAGCTAA    180                                                                 - - TTTTTTTATT TTTGGTAGAC ACGGGGTCTT GCTATGCTGC CCAGGCTGGT CT -            #CAAACTCC    240                                                                 - - CTGGGCTCAC GTGATCCTCC TGTCTCGGCC TCCCAAAGTG CTGGGATTAC AG -            #GTATGAAC    300                                                                 - - CACTGTGCCT GGCCCCACCC TGCACTTTGA AAGAGCACAG AGTGGGGTCA GG -            #GCCTGGCC    360                                                                 - - TGTGGGCATT AGGGCAGGTG TTTCACCGGG TTCTTGTTGA CCCATGCCAT GA -            #GATGGCCT    420                                                                 - - CAGTCATGCC AGTCCTACCT TCTGGGCCCA GGGTCCCCCT CATGGCTGCG TA -            #ACCTTGGG    480                                                                 - - CAAGTGGCTG AACCTCCCGG GCCTCACTTA TAAAACAAGC ATCATAATAG AA -            #CTGCAGCT    540                                                                 - - TGTGGCAGGA ATCACTAGAT TAAGGCACGC AAAGGGCTCA GTGCATTTGC CC -            #AAACCTGG    600                                                                 - - CCTTTGGTTG ACGTCCATAG CTTCAATTCG TATAAGGAAA ATATGGGGGC TA -            #CAGAAGGT    660                                                                 - - GGGGTCATAG ACCGTGGGGT TGCCCAAGCC AGGGGCGCTG TTGTCCATGT TT -            #CAGCAAAA    720                                                                 - - CAGATGTATT TTTCTCTGGG CGACAAAACT CATCCTAAAT GCGACTGAGA GC -            #CCTGTAAT    780                                                                 - - GTCCCAGGAC AGCTTGACCG CTGGGGTGGG TCCCCTTCCA CTGTCCCAGG CT -            #GGGGCGCT    840                                                                 - - GCGTCTGGGC TGCCCTTGGC ACCATCCACT CCTCTCTCGC CCACAGTCAA GT -            #ACGACCTT    900                                                                 - - AACAACCCGG CCAAGCACGC CAAGCTGGAC TTCCTCAACA ACCTGGCCAC GG -            #GCCTGGTG    960                                                                 - - TTCATCATCG TGGTAGTCAA CATCTTCATC ACGGCCTTCG GGGTCCAGAA GC -            #CCTTGATG   1020                                                                 - - GACATGGCAC CCCAGCAGTA GGACACCCAG GTGAGCTGGG AGATGGGGCG CG -            #AGGCCTGC   1080                                                                 - - AGTCCTGGGG TTGCTCGCTG TTGGAGGCTC TTGCAGTGTG GTGAGTCCCT GG -            #CCGGCCAG   1140                                                                 - - CCTTGGACAC CTTCCTAGGC CATGGGCATC CTCGTCCACA CCTACAAGGC CA -            #ATGCCTGG   1200                                                                 - - CCACTGCCTT GAGGCCAGCC CTGCCACTGG TGCTGGCCAC CTGGGGTCCT GT -            #GGTCACAG   1260                                                                 - - TGTTTAGATG GAATGTGTGT AGGAGCCACC ATTTGAACAT CCTGGAGAAC TC -            #ACTTAAAC   1320                                                                 - - GTAAGATTTC TATACATTCA GAATGTCTGT CCGATAAAAA AAAAAAAAAA AA - #AA             1374                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1001 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGCACGAGCG TGGCTCAAAC GACCGCCGCT AAGAACAAAA CGTTGGCTTT GG -             #CTTCGTTG     60                                                                 - - CAAAGCAGCC GCTCGGTGGC CGTACAACGC TTCATCTCTC CGAGCCTCGG TT -            #TCCTCATC    120                                                                 - - TCCAGCCCTA AAATGACGAC ACGCCCCACA GGTCTTGGGA GGATTAAGTG AG -            #GGGACATG    180                                                                 - - AGGTGGTCAT CGGTGTCCTG CTCGTGGTCA TTGGTGAGGA GCCCAGCCTG CA -            #GTCAGACC    240                                                                 - - TTCTGCCTCG GCACCCGTGG CTGGCAGAAA GGCCCCACGT GTCCCCTGGG CC -            #ACCCTGCA    300                                                                 - - TTGGCACAGG CAGCTTTGCA ACCACACGCT GACCTGCAGT GAGCCCTCCG CT -            #AACAGAGG    360                                                                 - - CCCAAAGACC AACTTCCACC CCGCGAGGGC AGGCGCCCTG TCCTGTCTCC TG -            #CACGGCTG    420                                                                 - - AACCTGAATG AGGTAGAAAA GCAGTGGCGA CTCAACCAGC TCAACAACGC AG -            #CCACCATC    480                                                                 - - TTGGTCTTCT TCACTGTGGT CATCAATGTT TTCATTACAG CCTTCGGGGC AC -            #ATAAAACA    540                                                                 - - GGGTTCCTGG CTGCCAGGGC CTCAAGGAAT CCTCTCTGAA TGCAGCCTGG GA -            #CCCAGGTT    600                                                                 - - CTGGGCCTGG AACTTCTGCC TCCTTCCTCC GTGATCTGCC AGGCTCGTGG GC -            #ACTTTCCA    660                                                                 - - CAGCCCAGGA GAGCTTCTGA AAGGACAGTA TAGCTGCCCT TGCTCCCTAC CC -            #ACAGCACC    720                                                                 - - TGAGTTAAAA AGTGATTTTT ATGTTATTGG TCTAAGGGAC TTCCATCTTG GT -            #CTGAAGTC    780                                                                 - - CTGAGCTCAG ACGCAGGTAC TGCCAGCCAT ACCTTCCTGG TAGCATCTGC TG -            #GACCTAAG    840                                                                 - - TAAGGCATGT CTGTCTAAGG CCAAGTCTGC CCGGCTTAAG GATGCTGGTT CT -            #GACTCTAC    900                                                                 - - CCCACTGCTT CCTTCTGCTC CAGGCCTCAA TTTTCCCTTC TTGTAAAATG GA -            #ATCTATAT    960                                                                 - - CTATAAAGGT TTCTTCAAAT CCAAAAAAAA AAAAAAAAAA A    - #                      - # 1001                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 907 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - ATCTCTCCGA GCCTCGGTTT CCTCATCTCC AGCCCTAAAA TGACGACACG CC -             #CCACAGGT     60                                                                 - - CTTGGGAGGA TTAAGTGAGG GGACATGAGC CTGGAAGCTC CGACCCCAGG AG -            #CCAGCCCA    120                                                                 - - TCAACCTGAA CCATTACGCC ACCAAGAAGA GCGTGGCGGA GAGCATGCTG GA -            #CGTGGCCC    180                                                                 - - TGTTCATGTC CAACGCCATG CGGCTGAAGG CGGTGCTGGA GCAGGGACCA TC -            #CTCTCATT    240                                                                 - - ACTACACCAC CCTGGTCACC CTCATCAGCC TCTCTCTGCT CCTGCAGGTG GT -            #CATCGGTG    300                                                                 - - TCCTGCTCGT GGTCATTGCA CGGCTGAACC TGAATGAGGT AGAAAAGCAG TG -            #GCGACTCA    360                                                                 - - ACCAGCTCAA CAACGCAGCC ACCATCTTGG TCTTCTTCAC TGTGGTCATC AA -            #TGTTTTCA    420                                                                 - - TTACAGCCTT CGGGGCACAT AAAACAGGGT TCCTGGCTGC CAGGGCCTCA AG -            #GAATCCTC    480                                                                 - - TCTGAATGCA GCCTGGGACC CAGGTTCTGG GCCTGGAACT TCTGCCTCCT TC -            #CTCCGTGA    540                                                                 - - TCTGCCAGGC TCGTGGGCAC TTTCCACAGC CCAGGAGAGC TTCTGAAAGG AC -            #AGTATAGC    600                                                                 - - TGCCCTTGCT CCCTACCCAC AGCACCTGAG TTAAAAAGTG ATTTTTATGT TA -            #TTGGTCTA    660                                                                 - - AGGGACTTCC ATCTTGGTCT GAAGTCCTGA GCTCAGACGC AGGTACTGCC AG -            #CCATACCT    720                                                                 - - TCCTGGTAGC ATCTGCTGGA CCTAAGTAAG GCATGTCTGT CTAAGGCCAA GT -            #CTGCCCGG    780                                                                 - - CTTAAGGATG CTGGTTCTGA CTCTACCCCA CTGCTTCCTT CTGCTCCAGG CC -            #TCAATTTT    840                                                                 - - CCCTTCTTGT AAAATGGAAT CTATATCTAT AAAGGTTTCT TCAAATCCAA AA -            #AAAAAAAA    900                                                                 - - AAAAAAA                 - #                  - #                       - #         907                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Met Asp Ser Gly Thr Glu Glu Tyr Glu Leu As - #n Gly Xaa Leu Xaa        Pro                                                                             1               5   - #                10  - #                15              - - Gly Xaa Pro Gly Ser Pro Asp Ala Ser Pro Xa - #a Arg Trp Gly Xaa Arg                  20      - #            25      - #            30                   - - Xaa Xaa Pro Ile Asn Val Asn His Tyr Ala Xa - #a Lys Lys Ser Ala Ala              35          - #        40          - #        45                       - - Glu Ser Met Leu Asp Ile Ala Leu Leu Met Al - #a Asn Ala Ser Gln Leu          50              - #    55              - #    60                           - - Lys Ala Val Val Glu Gln Gly Pro Ser Phe Al - #a Phe Tyr Val Pro Leu      65                  - #70                  - #75                  - #80        - - Val Val Leu Ile Ser Ile Ser Leu Val Leu Gl - #n Ile Gly Val Gly Val                      85  - #                90  - #                95               - - Leu Leu Ile Phe Leu Val Lys Tyr Asp Leu As - #n Asn Pro Xaa Lys His                  100      - #           105      - #           110                  - - Ala Lys Leu Asp Phe Leu Asn Asn Leu Ala Th - #r Gly Leu Val Phe Ile              115          - #       120          - #       125                      - - Ile Val Val Val Asn Ile Phe Ile Thr Ala Ph - #e Gly Val Gln Lys Pro          130              - #   135              - #   140                          - - Leu Met Asp Xaa Ala Pro Arg Gln                                          145                 1 - #50                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 100 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Met Asp Pro Gly Thr Glu Glu Tyr Glu Leu As - #n Gly Asp Leu Arg Pro      1               5   - #                10  - #                15               - - Gly Ser Pro Gly Ser Pro Asp Ala Ser Pro Pr - #o Arg Trp Gly Leu Arg                  20      - #            25      - #            30                   - - Asn Arg Pro Ile Asn Val Asn His Tyr Ala As - #n Lys Lys Ser Ala Ala              35          - #        40          - #        45                       - - Glu Ser Met Leu Asp Ile Ala Leu Leu Met Al - #a Asn Ala Ser Gln Leu          50              - #    55              - #    60                           - - Lys Ala Val Val Glu Gln Gly Asn Glu Phe Al - #a Phe Phe Val Pro Leu      65                  - #70                  - #75                  - #80        - - Val Val Leu Ile Ser Ile Ser Leu Val Leu Gl - #n Ile Gly Val Gly Val                      85  - #                90  - #                95               - - Leu Leu Ile Phe                                                                      100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Pro Pro Asn Trp Gly Leu Arg Asn Arg Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Pro Pro Arg Ala Gly Leu Arg Asn Arg Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Pro Pro Arg Trp Ala Leu Arg Asn Arg Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Pro Pro Arg Trp Gly Asn Arg Asn Arg Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Pro Pro Arg Trp Gly Leu Asn Asn Arg Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Pro Pro Arg Trp Gly Leu Arg Leu Arg Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Pro Pro Arg Trp Gly Leu Arg Asn Asn Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Pro Pro Arg Trp Gly Leu Arg Asn Arg Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Pro Arg Trp Gly Leu Arg Asn Arg Pro                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Arg Trp Gly Leu Arg Asn Arg                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Pro Ala Arg Trp Gly Trp Arg His Gly Pro Il - #e Asn                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Ala Arg Trp Gly Trp Arg His Gly Pro                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Arg Trp Gly Trp Arg His                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Arg Trp Gly Trp Arg                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - AGGGCAGGCA GCATCCAGGG TCCT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - TGTCCGGGTT GTTAAGGTCG TACTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - GAAGACCAAG ATGGTGGCTG CGTTG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - TTCAGGTTCA GCCGTGCAAT GACCA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - TCAGTCGCAT TTAGGATGAG TTTTG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - GGATGAGTTT TGTCGCCCAG AGAA          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Arg Trp Gly Trp Arg His Gly                                              1               5                                                            __________________________________________________________________________

We claim:
 1. A recombinant nucleic acid comprising a nucleic acidmolecule encoding a ninjurin protein having at least 70% homology to asequence selected from the group consisting of the sequence of FIG. 1B(SEQ ID NO: 4), FIG. 2B (SEQ ID NO: 6), and FIG. 3B (SEQ ID NO: 8).
 2. Arecombinant nucleic acid according to claim 1 wherein said ninjurinprotein is ninjurin
 1. 3. A recombinant nucleic acid according to claim1 wherein said ninjurin protein is ninjurin
 2. 4. A recombinant nucleicacid according to claim 1 wherein said ninjurin protein is a humanninjurin protein.
 5. A recombinant nucleic acid according to claim 1wherein said ninjurin protein has at least 85% homology to a sequenceselected from the group consisting of the sequence of FIG. 1B (SEQ IDNO: 4), FIG. 2B (SEQ ID NO: 6), and FIG. 3B (SEQ ID NO: 8).
 6. Arecombinant nucleic acid according to claim 1 wherein said ninjurinprotein has at least 90% homology to a sequence selected from the groupconsisting of the sequence of FIG. 1B (SEQ ID NO: 4), FIG. 2B (SEQ IDNO: 6), and FIG. 3B (SEQ ID NO: 8).
 7. A recombinant nucleic acidaccording to claim 1 wherein said ninjurin protein has at least 95%homology to a sequence selected from the group consisting of thesequence of FIG. 1B (SEQ ID NO: 4), FIG. 2B (SEQ ID NO: 6), and FIG. 3B(SEQ ID NO: 8).
 8. A host cell comprising the nucleic acid of claim 1.9. A recombinant nucleic acid encoding the amino acid sequence depictedin FIG. 1B (SEQ ID NO: 4).
 10. A recombinant nucleic acid encoding theamino acid sequence depicted in FIG. 2B (SEQ ID NO: 6).
 11. Arecombinant nucleic acid encoding the amino acid sequence depicted inFIG. 3B (SEQ ID NO: 8).
 12. A recombinant nucleic acid comprising thenucleic acid depicted in FIG. 1A (SEQ ID NO: 3).
 13. A recombinantnucleic acid comprising the nucleic acid depicted in FIG. 2A (SEQ ID NO:5).
 14. A recombinant nucleic acid comprising the nucleic acid depictedin FIG. 3A (SEQ ID NO: 7).
 15. A recombinant nucleic acid comprising thenucleic acid depicted in FIG. 4 (SEQ ID NO: 9).
 16. A recombinantnucleic acid comprising the nucleic acid depicted in FIG. 4 (SEQ ID NO:10).
 17. A recombinant nucleic acid comprising the nucleic acid depictedin FIG. 6 (SEQ ID NO: 11).